WO2019025608A1

WO2019025608A1 - Methods for treating leukaemia with an neuron-glial antigen 2 inhibitor

Info

Publication number: WO2019025608A1
Application number: PCT/EP2018/071177
Authority: WO
Inventors: Pablo MENÉNDEZ BUJÁN; Clara Bueno Uroz; Belén LÓPEZ MILLÁN; Heleia ROCA HO; Cristina PRIETO FERNÁNDEZ
Original assignee: Instituto De Investigación Contra La Leucemia Josep Carreras
Priority date: 2017-08-04
Filing date: 2018-08-03
Publication date: 2019-02-07

Abstract

The invention relates to a neuron-glial antigen 2 (NG2) inhibitor for use in the treatment of leukaemia. Additionally, the invention relates to in vitro methods for designing a customized therapy in a subject suffering from leukaemia based on determining the levels of NG2 and also to a method of determining whether a leukemic tumor is resistant or sensitive to chemotherapy based on determining the levels of NG2. The invention also relates to a combination comprising a neuron-glial antigen 2 (NG2) inhibitor and one or more therapeutic agents useful in the treatment of leukaemia.

Description

METHODS FOR TREATING LEUKAEMIA WITH AN NEURON-GLIAL ANTIGEN 2

INHIBITOR

TECHNICAL FIELD OF THE INVENTION The invention is related to the field of treatment of leukaemia.

BACKGROUND OF THE INVENTION

Leukaemia is a cancer of the blood or bone marrow characterized by the uncontrolled accumulation of blood cells, which is categorized into four types: acute lymphocytic leukaemia (ALL), acute myelogenous leukaemia (AML), chronic lymphocytic leukaemia (CLL), and chronic myelogenous leukaemia.

The bone marrow microenvironment provides a supportive environment for malignant hematopoietic cells, including leukaemias. This environment also provides protection from chemotherapeutic agents, potentially facilitating the survival of small numbers of residual cells that can ultimately lead to disease relapse.

Leukaemias arising from rearrangements of the mixed-lineage leukaemia (MLL) gene (MLLr leukaemias) make up approximately 10% of acute leukaemias in all age groups. For the most part, MLLr leukaemias are either acute lymphoid or acute myeloid leukaemias (ALL or AML, respectively). MLLr is associated with poor outcome in AML and B-ALL. MLL rearrangements are most commonly found in ALL in infants less than 12 months of age, but they are also found in older children or adults. MLLr as a subgroup of acute leukaemias is associated with certain phenotypic features that set it apart from other classes of leukaemias. MLLr acute leukaemias, particularly in infants, are more likely to present with hyperleukocytosis and CNS involvement. B-ALL blasts have the ability to cross the blood-cerebrospinal fluid (CSF) barrier, entering and seeding the CNS and, thus, producing CNS disease/relapse. In vitro, MLLr blasts often have resistance to commonly used chemotherapeutic drugs such as prednisone and L- asparaginase.

Of special interest is the infant B-ALL carrying MLLr (iMLLr-B-ALL), particularly the t(4;l 1)/MLL-AF4 (MA4), which results from the t(4;l I)(q21;q23), and represents a subtype of B-ALL with dismal prognosis. iMLLr-B-ALL has a distinctive pro-B/mixed phenotype (CD 10^" with expression of myeloid markers) and frequently shows therapy refractoriness and central nervous system (CNS) infiltration. Relapsed B-ALL is still common, and remains non curable to date.

The backbone of current induction or re -induction post-relapse treatment protocols for B-ALL comprises vincristine, glucocorticoids, and L-asparaginase (VxL), with or without an anthracycline (Szymanska B. et al. PloS One 2012, 7(3):e33894). Unfortunately, outcomes with conventional chemotherapy remain suboptimal to dismal for relapsed B-ALL.

Disruption of leukemic cell interactions with the bone marrow microenvironment has been postulated to be of therapeutic advantage. G-CSF has shown to enhance the efficacy of chemotherapy in patients with AML (Lowenberg B. et al. N Engl J Med, 349 (2003), pp. 743-752), but contradictory results have been obtained in other studies.

Therefore, there is a clear and unmet need for effective therapeutics for treatment of leukaemias, particularly MLLr-B-ALL, to overcome therapy resistance, relapse and CNS disease.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a neuron-glial antigen 2 (NG2) inhibitor for use in the treatment of leukaemia.

In a second aspect, the invention relates to an in vitro method for designing a customized therapy for a subject diagnosed with leukaemia which comprises:

a) determining the levels of NG2-expressing cells in a sample from said subject, and

b) comparing said levels with a reference value

wherein increased levels of NG2-expressing cells with respect to the reference value are indicative that the subject is to be treated with a neuron-glial antigen 2 (NG2) inhibitor.

In a third aspect, the invention relates to an in vitro method for determining whether a tumor is resistant or sensitive to chemotherapy in a subject suffering from leukaemia comprising: a) determining the levels of NG2-expressing cells in a sample from said subject, and

b) comparing said levels with a reference value,

wherein increased levels of NG2-expressing cells with respect to the reference value are indicative that the tumor is resistant to chemotherapy, and decreased levels of NG2- expressing cells with respect to the reference value are indicative that the tumor is sensitive to chemotherapy.

In a fourth aspect, the invention relates to a combination comprising a neuron- glial antigen 2 (NG2) inhibitor and one or more therapeutic agents useful in the treatment of leukaemia.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Clinical impact of NG2 expression in MLLr infant B-ALL. A) The hazard ratio (HR) of relapse for different cut-offs of NG2 expression was investigated to define NG2^high versus NG2^low patients (n=55). The HR of 1.75 corresponding to the 40% cut-off was used. B) Five-year event free survival (EFS) in NG2^high and NG2^low patients. C) Frequency of patients with immature CD10^neg immunophenotype in NG2^high and NG2^low subgroups (left panel), White blood cell (WBC) count at diagnostic (middle panel) and frequency of CNS disease (right panel) in NG2^hlgh and NG2^l0W patients.

Figure 2. Leukemia development and phenotype in primografts of NG2+ and NG2- blast populations. A) Representative NG2 immunophenotype of diagnostic MA4⁺ BM samples, and high-purity FACS-sorting of NG2⁺ and NG2^" populations. More than 95% of NG2⁺ and NG2^" blasts carry the t(4; 1 1)/MA4 by FISH. B) Outline of the in vivo experimental design. NG2⁺ or NG2^" blasts were IBM-transplanted into NSG mice at day 0. The health of the mice and leukemia development were monitored over 20 weeks. Mice were sacrificed when disease was evident, when leukemic cells were 10% in PB, or at day 140 (in the absence of symptoms or PB engraftment). C) Kaplan- Meier survival curves for event-free survival (EFS) according to decreasing cell doses (200k to lk) for NG2 and NG2^" transplanted mice. D) Estimated frequency (and 95% confidence interval) of L-IC in NG2⁺ and NG2^" primografts calculated for cell doses <200k or <50k. E) Representative flow cytometry analysis of leukemic mice. The human graft, identified as CD45⁺HLA-ABC⁺, reproduces the pro-B phenotype (CD34 CD19 CD10 ) seen in patients. Engrafted leukemias always re-establish NG2 variable expression and carry the t(4;l 1)/MA4 as detected by dual-fusion or break-apart FISH. F) Level of leukemia engraftment in hematopoietic tissues from mice transplanted with NG2⁺ and NG2^" blasts. Injected (IT) and contralateral (CL) tibia, liver, spleen and peripheral blood (PB). Each dot represents a transplanted mouse and bars represent mean level of engraftment. G) Both NG2⁺ and NG2^" transplanted mice consistently displayed splenomegaly, high WBC counts, and a skewed granulocytic to lymphoid cell representation in PB. Control group includes non-engrafted mice. *p<0.05

Figure 3. NG2 expression does not enrich for L-IC capacity in secondary recipients. A) Top panels, Kaplan-Meier survival curves for EFS according to different cell doses transplanted into secondary recipients. Black and grey lines represent secondary recipients transplanted with NG2+ and NG2- primary animals, respectively. Dotted line depicts EFS rate of 50%. Bottom panels, Representative immunophenotype of leukemias in secondary mice. The human graft, identified as CD45+ and HLA.ABC+, reproduces the phenotype seen in the primary leukemia and primary recipients: CD34+CD19+CD10- immature B-lymphoid cells with variable expression of NG2. B) Percentage of long-term leukemic engraftment in the injected (IT) and contralateral (CL) tibia, liver, spleen and PB of secondary mice. C) Secondary recipients of cells from either NG2+ or NG2- primary mice consistently displayed splenomegaly, high WBC counts and a skewed granulocytic to lymphoid cell representation in PB. Control mice are non-engrafted mice.* p<0.05

Figure 4. NG2 expression is up-regulated in extramedullary hematopoietic tissues. A) Percentage of NG2-expressing blasts (black bar) in primary (top panels) and secondary (bottom panels) engrafted mice at sacrifice. The following tissues were analyzed: IT, intra-tibia; CL, contralateral tibia; Liv, liver; Sp, spleen; PB, peripheral blood. Left panels, NG2+ mice. Right panels, NG2- mice. The initial condition represents the percentage of NG2-expressing cells at the moment of transplantation: 100% for primary NG2+ mice (black bar) and 0% for primary NG2- mice (gray bar). B) Left panel, RT-qPCR confirming 7-fold-higher expression of NG2 in xenografted liver as compared to BM. Right panel, NG2+ and NG2- cells were sorted and IV injected. Mice were weekly monitored by FACS for chimerism and sacrificed after 7-8 weeks. Leukemia engraftment in PB is shown for NG2+ (black dots) and NG2- (grey dots) transplanted mice. C) Three-fold-higher blast cell counts in diagnostic NG2^hlgh versus NG2^l0W MLL-AF4+ infants. Mean of NG2+ cells was used as cut-off (Table 1). *p<0.05.

Figure 5. NG2 is not a prospective marker for CNS-IC but it is expressed in almost all MLLr blasts entering the CNS. A) Top panels, representative H&E staining of mice brains defining xenografts with negative and positive CNS involvement. S, skull. P, brain parenchyma. Leukemic infiltration is exclusively found in leptomeninges and is marked by a white arrowhead. The right panel (macro) depicts the area magnified on each row. Bottom panels, H&E staining and immunohistochemistry for CD 19 and CD45 performed on paraffin-embedded skulls from mice transplanted with NG2+ and NG2- blasts. Chimeric spleens and skulls from non-engrafted mice were used as positive and negative controls, respectively. CD45+CD19+ human blasts are marked with a white arrowhead. B) Top panel, number (and percentage) of patients showing CNS infiltration at diagnosis (Y-axis) vs number (and percentage) of patient samples developing CNS disease in mice (X-axis). Bottom panel, percentage of mice displaying CNS involvement according to NG2 phenotype of transplanted blasts. C) RT-qPCR showing 55 -fold-higher expression of NG2 in spinal cord than in BM. *p<0.05, n.s. not significant.

Figure 6. Mice transplantation and treatment with chondrotinase (Ch'ase) and an anti-NG2 monoclonal antibody (7.1 MoAb). For intravenous (iv) transplantation NG2- or NG2+ sorted blasts were transplanted via the lateral tail vein. PB was collected weekly to analyze leukemia engraftment by flow cytometry. Once leukemia engraftment reached 10% in PB, mice were treated intraperitoneally (i.p.) either with Chase (Ch'ase 0.06 U/mouse daily or with 7.1 MoAb 10 mg/kg daily for 7 days and then sacrificed. BM, bone marrow; PB, peripheral blood.

Figure 7. Engraftment of NG2+ leukemic cells. NG2+ and NG2- cells were sorted and IV injected. Mice were sacrificed after 4 weeks. Leukemia engraftment in peripheral blood is shown for NG2+ (black dots) and NG2- (black squares) transplanted mice. Figure 8. Blocking of engraftment after treatment with inhibitors of NG2.

NG2+ blasts were preincubated with Chase (CHASE), and with two anti-NG2 antibodies: 7.1 clone and 9.2.21 clone. Control without treatment (NO TREATMENT).

Figure 9. Blast expression of NG2 before (ctrol) and after the in vitro treatment with Chase (Ch'ase) analyzed by FACS.

Figure 10. Blast expression of NG2 before (ctrol) and after the in vivo treatment with Chase (Ch'ase) and monoclonal anti-NG2 antibody (7.1) analyzed by FACS. PB, peripheral blood. BM, bone marrow.

Figure 11. The leukemic engraftment decreases in BM and increases in PB after in vivo treatment with NG2 inhibitors. A) Percentage of leukemic cells in BM and PB referred to day 0 (DO) after 7 days of treatment (D7) with Chase or monoclonal anti-NG2 antibody (7.1). B) Ratio of leukemic cells in PB/BM after 7 days of treatment or without treatment. BM, bone marrow. PB, peripheral blood.

Figure 12. NG2+ cells are more resistant to chemotherapy than NG2- cells. NG2+ and NG2- blasts were in vitro incubated with dexamethasone (A) or with a combination of dexamethasone, L-asparaginase and vincristine (B) and the percentage of alive cells was measured.

Figure 13. Pre-treatment with chondroitinase enhances the cytotoxicity of chemotherapy in vivo. Mice were either left untreated (CTROL) or treated with a combination of vincristine, L-asparaginase and dexamethasone (VxL) after a pretreatment with chondroitinase. Percentage of cells positive for the anti-NG2 antibody 7.1 in peripheral blood with respect to the total number of blasts (NG2+ and NG2-) was obtained.

Figure 14. In vivo blockage of NG2 results in a robust mobilization of MLLr-B-ALL blasts into PB. A) Monitoring levels of leukemic graft in PB and BM before (day 0) and after (day 7) the indicated treatments. Each line represents the same mouse before and after treatment. B) Levels of leukemic graft in BM (top panel) and PB (bottom panel) after the indicated in vivo treatments. Results are shown as mean±SEM, relative to day 0 (before treatment). C) Representative FACS plots showing identical leukemia NG2+ phenotype in both diagnostic sample and primografts. The right panel shows the in vivo effectiveness of the 7.1 MoAb which abolishes NG2 expression in blasts recovered from primografts. D) BM-MSCs protect MLLr-B-ALL cells against VxL chemotherapy. *p<0.05; **p<0.01; ****p<0.0001.

Figure 15. In vivo treatment with ch'ase sensitizes blasts to VxL rendering higher CR rates and higher EFS in pre-clinical PDX models of MLLr-B-ALL. A) Complete experimental design of the pre-clinical PDX models detailing in vivo treatments with VxL and NG2 inhibitors. After 2 complete cycles of VxL chemotherapy, MRD/CR was evaluated and relapse was followed up for up to 35 days. B) Monitoring levels of leukemic graft in PB and BM for the indicated treatments. PB engraftment was analyzed weekly. BM leukemic engraftment was analyzed by BM aspirates at the end of VxL±Ch'ase treatment (day 15) and at the end of follow-up period (day 50). Each line represents the same mouse before and after treatment. C) Levels of leukemia engraftment in BM at treatment initiation (day 0) for VxL or VxL+Ch'ase mice cohorts. D) BM levels of MRD at the end of 2 cycles of VxL±Ch'ase (day 15). Each dot represents a single mouse. A mouse is considered in CR when the % of blasts in BM<1% (horizontal dotted line). The light bars represent the proportion of mice in CR (right Y-axis) for VxL and VxL+Ch'ase. E) Kaplan-Meier survival curves for 45-days EFS (n=16 mice in each group). F) Leukemic burden in BM at sacrifice. *p <0.05; n.s.: no significant differences.

Figure 16. In vivo treatment with 7.1 MoAb sensitizes blasts to VxL providing higher CR rates and higher EFS in pre-clinical PDX models of MLLr-B- ALL. A) Monitoring levels of leukemic graft in PB and BM for the indicated treatments. PB engraftment was analyzed weekly. BM leukemic engraftment was analyzed by BM aspirates at the end of VxL±7.1 MoAb treatment (day 15) and at the end of follow-up period (day 50). Each line represents the same mouse before and after treatment. B) Levels of leukemia engraftment in BM at treatment initiation (day 0) for VxL or VxL±7.1 MoAb mice cohorts. C) BM levels of MRD at the end of 2 cycles of VxL±7.1 MoAb (day 15). Each dot represents a single mouse. A mouse is considered in CR when the % of blasts in BM<1% (horizontal dotted line). The light bars represent the proportion of mice in CR (right Y-axis) for VxL and VxL±7.1 MoAb. D) Kaplan- Meier survival curves for 45-days EFS (n=10-l 1 mice in each group). *p <0.05; n.s.: no significant differences. DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have surprisingly found that blocking NG2 prevents the engraftment of a leukaemia in bone marrow (Example 6) and that the in vivo administration of an inhibitor of NG2 after the leukaemia is engrafted causes that blasts lose their capability to anchor in bone marrow and migrate to peripheral blood (Example 7).

Particularly, the inventors of the present invention have shown that iMLLr-B- ALL NG2+ blasts pretreated with chondroitinase or monoclonal antibodies against NG2 are not capable of migrating to the bone marrow and engraft there (Example 6). Furthermore, the in vivo treatment of mice having active iMLLr-B-ALL in bone marrow and peripheral blood with chondroitinase or monoclonal antibodies against NG2 produces a decrease in the leukemic engraftment in bone marrow and an increase of the engraftment in peripheral blood (Example 7). Additionally, the inventors have shown that blocking NG2 synergyzes with established current induction therapy for B-ALL based on vincristine, glucocorticoids, and L-asparaginase (VxL) backbone (Example 10). When combined with NG2 inhibitors (chondroitinase or monoclonal antibodies against NG2), VxL treatment achieves higher rates of complete remission, and consequently higher EFS and delayed time-to-relapse.

These findings support the use of inhibitors of NG2 to decrease the tumoral mass in bone marrow and promote the migration of leukemic cells from bone marrow to peripheral blood thus increasing the availability of said cells to respond to chemotherapy.

Furthermore, the inventors have shown that the expression of NG2 in a tumor sample from a subject suffering leukaemia may be used as an indicator of the response of said tumor to a chemotherapeutic treatment (Example 8).

Medical uses The authors of the present invention have found that NG2 inhibitors are therapeutically effective in the treatment of leukaemias. In a first aspect, the invention relates to a neuron-glial antigen 2 (NG2) inhibitor for use in the treatment of leukaemia.

Alternatively, the invention relates to the use of a neuron-glial antigen 2 (NG2) inhibitor for the preparation of a medicament for the treatment of leukaemia.

Alternatively, the invention relates to a method for treating leukaemia comprising administering a neuron-glial antigen 2 (NG2) inhibitor to a subject in need thereof.

"Neuron-glial antigen 2" or "NG2" or "chondroitin sulfate proteoglycan 4", also known as CSPG4 in humans, as used herein, is a cell surface type I transmembrane proteoglycan not expressed in normal hematopoietic cells. By contrast, l lq23/MLLr leukemias specifically express NG2. This protein is covalently modified with chondroitin sulfate glycosaminoglycan and harbors a large ectodomain composed of three subdomains. The N-terminal domain (Dl subdomain) contains two laminin-like globular (LG) repeats. It is likely that the LG domains as in other proteoglycans mediate ligand binding, cell-matrix and cell-cell interactions, as well as interaction with integrins and receptor tyrosine kinase (RTK). The central subdomain D2 contains 15 tandem repeats of a new module called CSPG. The CSPG repeat is a cadherin-like and tumor-relevant module which is predicted to be involved in cell-matrix interaction, further modulated by the chondroitin sulfate chain covalently attached to this module. Indeed, CSPG modules bind to collagens V and VI, FGF and PDGF. The juxtamembrane subdomain D3 contains a carbohydrate modification able to bind integrins and galectin, as well as numerous protease cleavage sites. The transmembrane domain of NG2 has a unique Cys residue, generally not found in transmembrane regions. The intracellular domain harbors a proximal region with numerous Thr phospho-acceptor sites for PKCa and ERK1/2, and a distal region encompassing a PDZ-binding module similar to the syndecan family. The latter can bind to the PDZ domain of several scaffold proteins involved in intracellular signaling, including syntenin, MUPP1 and GRIP1. The complete protein sequence for human NG2 has the UniProt accession number Q6UVK1 (June 7, 2017). The NG2 protein has different names designating the same gene product in different species. For example, the term CSPG4 designates the human gene product whereas the rat ortholog is called NG2. In the context of the present invention, the term "NG2" encompasses all orthologs of the CSPG4 human protein.

The term "inhibitor", as used herein when referred to NG2, includes without limitation, compounds that bind to the extracellular domains of NG2 or physically interact with them such as antagonists of NG2, or antibodies against the NG2 proteoglycan; compounds capable of degrading proteoglycans, particularly capable of degrading chondroitin sulfate glycosaminoglycans; compounds which prevent the binding of NG2 to its natural ligands; compounds which prevent expression of the NG2 proteoglycan and compounds which lead to reduced mRNA or protein levels of NG2 proteoglycan. The term "inhibitor" refers preferably to a compound capable of binding to NG2, more preferably capable of preventing NG2 glycoprotein from binding to one or more of its natural ligands, for example because the compound itself binds to NG2 masking part of its structure, avoiding that its ligands bind to it. Compounds capable of binding to NG2 can be determined by binding assays well known by a person skilled in the art. Binding assays can also be used to assess the NG2 binding to its natural ligands such as, for example, those assays disclosed in Burg M.A. et al. J. Biol. Chem. 1996; 271(42):26110-6). Exemplary NG2 natural ligands that can be assayed are, without limitation, integrins, receptor tyrosine kinase, collagens V and VI, FGF, PDGF, galectin, angiostatin, and plasminogen. The term "inhibitor" also refers preferably to a compound that prevents or reduces the expression of NG2 mRNA or NG2 protein; or to a compound that degrades part of the NG2 structure, preferably avoiding that its ligands bind to NG2.

The person skilled in the art knows how to determine if a particular molecule is an inhibitor of said proteoglycan. In addition to binding assays, an inhibitor useful for the present invention may be identified by the method disclosed in Examples 6 and 7 of the present patent application wherein the expression of NG2 in blasts is assayed by flow cytometry with an anti-NG2 antibody before and after the incubation with or the administration of the compound. The compound assayed is an inhibitor of NG2 when NG2 expression is detected in the membrane of blasts before the incubation with or the administration of the compound but cannot be detected after the blasts have been incubated with the compound or after the compound has been administered. The NG2 expression can be detected with the 7.1 anti-NG2 monoclonal antibody (Beckman Coulter).

The NG2 inhibitors can be, among others, proteins, peptides, interference RNA, antisense oligonucleotides or small organic molecules.

In an embodiment the inhibitor is an antagonist. In the context of the present invention, the term "antagonist" refers to a compound that binds to NG2 and lacks any substantial ability to activate it. An antagonist can thereby prevent or reduce the functional activation of NG2 by its natural ligand.

In a preferred embodiment, the inhibitor is a proteoglycan-degrading enzyme. The term "proteoglycan-degrading enzyme", as used herein, refers to an enzyme capable of catabolizing proteoglycans and, particularly, capable of catabolizing NG2. Exemplary proteoglycan-degrading enzymes can be, without limitation, proteinases (such as cathepsin F, cathepsin Bl, cathepsin D, papain, pronase, trypsin), chondroitinases or sulphatases. In a preferred embodiment, the proteoglycan-degrading enzyme is an enzyme that hydro lyzes glycosyl bonds, preferably is a chondroitin sulfate glycosaminoglycan degrading enzyme, more preferably chondroitinase. The term "chondroitinase" refers to a class of enzymes that catalyse the hydrolysis of chondroitin sulfate proteoglycans. The chondroitinase useful in the present invention may be, without limitation, N-acetylgalactosamine-4-sulfatase, N-acetylgalactosamine-6- sulfatase, chondroitin lyase AC I (EC 4.2.2.5), chondroitin lyase AC II (EC 4.2.2.5), chondroitin B lyase (EC 4.2.2.19), or chondroitin ABC lyase (EC 4.2.2.4); preferably chondroitin ABC lyase also named Chase ABC (EC 4.2.2.4).

The term "chondroitin ABC lyase", as used herein, refers to the enzyme EC 4.2.2.4, which is a mixture of ABC lyases I and II and acts on CS-A, CS-B and CS-C in a predominantly endolytic action pattern. Preferably, the chondroitin ABC lyase is from P. vulgaris.

The activity of a proteoglycan-degrading enzyme can be assayed by measuring the quantity of proteoglycan that has been degraded by the enzyme or by detecting the absence of the proteoglycan after the degradation. The assay depends on the specific enzyme and proteoglycan involved. Exemplary methods for detecting degradation of a proteoglycan are disclosed in Dingle J.T. et al. Biochem J. 1977; 167:775-785. When the proteoglycan-degrading enzyme is a chondroitin sulfate degrading enzyme, the degradation of chondroitin sulfate can be assessed by incubation with an antibody that recognizes chondroitin sulfate, said incubation carried out before and after the treatment with the enzyme. The antibody that recognizes chondroitin sulfate can be, for example, the monoclonal antibody anti CS [chondroitin sulfate] 2B6 (Amsbio).

In a preferred embodiment, the inhibitor is an antibody capable of binding to

NG2. Assays for assessing if an antibody is capable of binding to its antigen are well known by those skilled in the art. Preferably, the antibody is an inhibitory antibody. The term "inhibitory antibody" is understood to mean, according to the present invention, an antibody that is capable of binding to NG2 provoking the inhibition of the activation of this proteoglycan by its natural ligands or that is capable of binding to NG2 preventing that NG2 binds to one or more of its natural ligands. Antibodies may be prepared using any method known by a person skilled in the art. Thus, polyclonal antibodies are prepared by immunization of an animal with the protein aimed to be inhibited. Monoclonal antibodies may be prepared using the method described by Kohler, Milstein et al (Nature, 1975, 256: 495). Once antibodies capable of binding to NG2 are identified, those antibodies capable of inhibiting NG2 activity using the abovementioned assays for determination of NG2 activity will be selected. Suitable antibodies in the present invention include intact antibodies which comprise an antigen- binding variable region and a constant region, fragments "Fab", "F(ab')2", "Fab"', Fv, scFv, diabodies and bispecific antibodies. In a preferred embodiment, the antibody is clone 7.1 anti-NG2 monoclonal antibody (Beckman Coulter). In another embodiment, the antibody is an antibody binding to the same epitope than clone 7.1 anti-NG2 monoclonal antibody (Beckman Coulter). Antibodies binding to the same epitope than the antibody used in the experimental part of this patent application can be found by epitope competition assays for their antigen binding with clone 7.1 anti-NG2 monoclonal antibody.

In another embodiment, the inhibitor is an interference RNA. As used herein, the term "interference RNA" or "iRNA" refers to RNA molecules capable of silencing the expression of NG2 gene or of any gene needed for NG2 function. To that end, iRNA are typically double-stranded oligonucleotides having at least 30 base pairs in length, and they more preferably comprise about 25, 24, 23, 22, 21, 20, 19, 18 or 17 ribonucleic acid base pairs. Several different types of molecules have been used effectively in iRNA technology including small interfering RNA (siRNA) sometimes known as short interference RNA or silencer RNA, micro RNA (miRNA) which normally differ from siRNA because they are processed from single-stranded RNA precursors and they are shown to be only partially complementary to the target mRNA and short hairpin RNA (shRNA).

Small interfering RNA (siRNA) agents are capable of inhibiting target gene expression by interfering RNA. siRNAs may be chemically synthesized, or may be obtained by in vitro transcription, o may be synthesized in vivo in target cell. Typically, siRNAs consist of a double-stranded RNA from 15 to 40 nucleotides in length and may contain a protuberant region 3' and/or 5' from 1 to 6 nucleotides in length. Length of protuberant region is independent from total length of siRNA molecule. siRNAs act by post-transcriptional degradation or silencing of target messenger.

siRNA may be denominated shRNA (short hairpin RNA) characterized in that the antiparallel strands that form siRNA are connected by a loop or hairpin region. siRNAs are constituted by a short antisense sequence (19 to 25 nucleotides) followed by a loop of 5-9 nucleotides, and the sense strand. shRNAs may be encoded by plasmids or virus, particularly retrovirus and, more particularly, retrovirus and under the control of promoters such as U6 promoter for RNA polymerase III.

The siRNAs of the invention are substantially homologous to NG2 mRNA or this protein-coding genome sequence. The term "substantially honomogous" is understood to mean that siRNAs have a sequence sufficiently complementary or similar to target mRNA so that siRNA may be able to provoke mRNA degradation by RNA interference. Suitable siRNAs to provoke interference include siRNAs formed by RNA, as well as siRNAs containing chemically different modifications such as:

-siRNAs in which the links between nucleotides are different from those appearing in nature, such as phosphorothioate links.

-Stranded-RNA conjugates with a functional reagent, such as a fluorophoro. -Modification of the ends of RNA strands, particularly the 3' end by the combination with different functional hydroxyl groups at 2 '-position.

-Sugar-modified nucleotides such as O-alkylated radicals at 2'-position such as

2'-0-methylribose or 2'-0-fluororibose. -Base-modified nucleotides such as halogenated bases (for example, 5- bromouracil and 5-iodouracil) or alkylated bases (for example, 7-methyl- guanosine).

The siRNAs and shRNAs of the invention may be obtained using a series of techniques known to a person skilled in the art. For example, siRNA may be chemically synthesized from protected ribonucleoside phosphoramidites in a conventional DNA/RNA synthesizer. Alternatively, siRNA may be produced by recombinant dicer from plasmid and viral vectors, where the coding region of siRNA strand or strands is under operative control of RNA polymerase III promoters. RNase Dicer processes shRNA into siRNA in cells .

The region which is taken as a basis for the design of siRNA is not limitative and may contain a region of coding sequence (between the initiation codon and the termination codon) or, alternatively, may contain sequences from the 5' or 3' untranslated region, preferably from 25 to 50 nucleotides in length and in any position in 3' position with regard to the initiation codon. A procedure for siRNA design involves the identification of sequence motive AA(N19)TT wherein N may be any nucleotide in the sequence of interest and the selection of those that exhibit a high content in G/C. If said sequence motive is not found, it is possible to identify sequence motive NA(N21) wherein N may be any nucleotide.

In another embodiment, the inhibitor is an antisense oligonucleotide, i.e. molecules whose sequence is complementary to mRNA coding for NG2, i.e., complementary to cDNA coding strand. The antisense oligonucleotide may be complementary to a complete coding region or a region of same including both the coding region and the 5' and 3' untranslated regions. The antisense oligonucleotides may consist of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. The antisense oligonucleotides may be obtained by chemical synthesis or by enzymatic binding reactions widely known to a person skilled in the art. For example, an antisense oligonucleotide may further contain modified nucleotides which increase its biological stability or the stability of the bicatenary DNA-RNA complexes formed between the antisense oligonucleotide and the target polynucleotide, such as phosphorothioate derivatives, peptide nucleic acids and acridine-substituted oligonucleotides. Modified oligonucleotides that may be used for the preparation of antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetyl-citosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethyl-aminomethyl uracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcitosine, 5- methylcitosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5 -methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2- thiocitosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3- amino-3-N-2-carboxypropyl)uracil, and 2,6- diaminopurine. Alternatively, the antisense nucleic acid may be produced biologically using an expression vector in which the antisense-oriented nucleic acid has been cloned.

In another embodiment, the inhibitor is a ribozyme or DNA enzyme. Ribozimes comprise a catalytic region and a second region whose sequence is complementary to target nucleic acid and confers substrate specificity on the ribozyme. After the interaction between the ribozyme and its substrate by hybridization and coupling between complementary regions of target nucleic acid and ribozyme, an activation of the catalytic region is produced provoking the inter- or intramolecular rupture of target nucleic acid. Basic considerations for the design of ribozymes are widely known to a person skilled in the art (see, for example, Doherty and Doudna (Annu. Rev. Biophys. Biomol. Struct. 2001; 30:457- 75).

Other compounds capable of inhibiting NG2 that can be used in the invention include aptamers and spiegelmers. Aptamers and spiegelmers are single -stranded or double-stranded D- or L-nucleic acids that specifically bind to the protein resulting in a modification of the biological activity of the protein. Aptamers and spiegelmers are 15 to 80 nucleotides in length and, preferably, 20 to 50 nucleotides in length.

Suitable methods for determining whether an inhibitor is capable of decreasing mRNA levels include, without limitation, standard assays for determining mRNA expression levels such as qPCR, RT-PCR, RNA protection analysis, Northern blot, RNA dot blot, in situ hybridization, microarray technology, tag based methods such as serial analysis of gene expression (SAGE) including variants such as LongSAGE and SuperSAGE, microarrays, fluorescence in situ hybridization (FISH), including variants such as Flow-FISH, qFiSH and double fusion FISH (D-FISH), and the like.

Suitable methods for determining whether an inhibitor acts by decreasing the NG2 protein levels include the quantification by means of conventional methods, for example, using antibodies with a capacity to specifically bind to the proteins encoded by the gene (or to fragments thereof containing antigenic determinants) and subsequent quantification of the resulting antibody-antigen complexes.

In another embodiment, the inhibitor is a small organic molecule or a pharmaceutically acceptable salt thereof.

The term "pharmaceutically acceptable salt thereof, as used herein, refers to derivatives of small organic molecules wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from nontoxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 1 ,2-ethanedisulfonic, 2-acetoxybenzoic, 2-hydroxyethanesulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodide, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methanesulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicylic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, and toluenesulfonic.

The pharmaceutically acceptable salts of the compounds can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are useful. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa., 1990, p. 1445.

An inhibitor of the invention may inhibit NG2 expression, NG2 detection of expression or NG2 binding to one of its natural ligands by at least 5%, at least 10%, at least 25%, at least 50%>, at least 75%, or at least 90%, and all ranges between 5% and 100%. Suitable methods for determining whether an inhibitor acts by decreasing the NG2 expression, NG2 detection of expression or NG2 binding to one of its natural ligands have been previously described.

In a preferred embodiment, the NG2 inhibitor is selected from the group consisting of chondroitinase, an antibody, interference RNA, an antisense oligonucleotide, a ribozyme, an aptamer and an spiegelmer; preferably is selected from the group consisting of an antibody and chondroitinase.

In an embodiment, if the leukaemia is a MLLr, more preferably a MLLr ALL, even more preferably a iMLLr-B-ALL, the NG2 inhibitor is an anti-CSPG4 chimeric antigen receptor, preferably is an immune effector cell comprising a CAR, more preferably is a engineered T-cell (CAR-T).

In another embodiment, the NG2 inhibitor is an antibody that does not form part of a chimeric antigen receptor (CAR), preferably that does not form part of an engineered T-cell (CAR-T).

According to the invention, the NG2 inhibitor is useful for treating a subject suffering leukaemia. In a preferred embodiment of the invention, the subject is a mammal. In a more preferred embodiment of the invention, the subject is a human of any race and sex.

The term "treatment", as used herein, means achieving a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit relates to the administration of an inhibitor according to the invention or of a medicament comprising said inhibitor to a subject suffering from a leukaemia including the administration in an initial or early stage of a disease, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treatment also means prolonging survival as compared to expected survival if not receiving the treatment. For prophylactic benefit, the inhibitor may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made. The term "treatment" within the context of the present invention also includes preventing the relapse of leukaemia or improving the response to chemotherapy.

As the skilled person acknowledges, effectiveness of a NG2 inhibitor in a therapy may be demonstrated by analyzing the haematological response (measure the numbers of white cells, red cells and platelets and the levels of hemoglobin and hematocrit), cytogenetic response and/or serological tumor markers.

Even though individual needs vary, determination of optimal ranges for therapeutically effective amounts of the inhibitor for use according to the invention belongs to the common experience of those experts in the art. In general, the dosage needed to provide an effective treatment, which can be adjusted by one expert in the art, will vary depending on age, health, fitness, sex, diet, weight, degree of alteration of the receptor, frequency of treatment, nature and condition of the injury, nature and extent of impairment or illness, medical condition of the subject, route of administration, pharmacological considerations such as activity, efficacy, pharmacokinetic and toxicology profile of the particular compound used, if using a system drug delivery, and if the inhibitor is administered as part of a combination of drugs.

The inhibitor of the invention may be administered by any suitable administration route, such as, but not limited to, parenteral, oral, topical, nasal, rectal route. In a particular embodiment, the inhibitor described herein is administered by parenteral route, e.g. by intravenous, intrathecal, intraperitoneal, subcutaneous, intradermal, intramuscular or epidural administration. In a preferred embodiment, the inhibitor is administered by intraperitoneal route. In another preferred embodiment, the inhibitor is administered by intrathecal route.

"Leukaemia", as used herein, refers to a type of cancer of the blood or bone marrow characterized by an abnormal increase of immature white blood cells called "blasts". By "blast cells" and "blasts" it is meant the immature precursors of lymphocytes (lymphoblasts), granulocytes (myeloblasts), monocytes (monoblasts), thrombocytes (megacaryoblasts) or erythrocytes (proerythroblasts). Blast cells can be recognized by their large size and primitive nuclei (i.e. the nuclei contain nucleoli). In addition, they may be identified and isolated from a blood sample by flow cytometry based on their mid-level expression of the cell surface marker CD45 and their high Side Scatter (SSC) activity. Leukaemia is a broad term covering a spectrum of diseases. In turn, it is part of the even broader group of diseases affecting the blood, bone marrow, and lymphoid system, which are all known as haemato logical neoplasms. There are four major kinds of leukaemia: Acute lymphoblastic leukaemia, or ALL; Acute myeloid leukaemia, or AML; Chronic lymphocytic leukaemia, or CLL; Chronic myelogenous leukaemia, or CML.

In a preferred embodiment the leukaemia to be treated is an acute leukaemia, preferably selected from ALL and AML.

The term "acute leukaemia", as used herein, refers to a rapidly progressive leukaemia if not treated and involves more immature cells. The term B-cell acute leukaemia refers to an acute leukaemia in which immature B-cells are found.

"Acute lymphoblastic leukaemia (ALL) or acute lymphoid leukaemia" is an acute form of leukaemia, or cancer of the white blood cells, characterized by the overproduction of cancerous, immature white blood cells— known as lymphoblasts.

"Acute Myeloid Leukaemia (AML) or acute myelogenous leukaemia or acute nonlymphocytic leukaemia (ANLL)" is a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal white blood cells (mieloblasts) that accumulate in the bone marrow and interfere with the production of normal blood cells. The symptoms of AML are caused by replacement of normal bone marrow with leukaemic cells, which causes a drop in red blood cells, platelets and normal white blood cells. The combination of a myeloperoxidase or Sudan black stain and a nonspecific esterase stain on blood and blood marrow smears are helpful in distinguishing AML from ALL.

In another embodiment, the leukaemia to be treated is a plasmacytoid dendritic cell (pDC) leukaemia. The term "plasmacytoid dendritic cell leukaemia" or "pDC" refers to a leukaemia classified as AML and related precursor neoplasms, that is a blastic plasmacytoid dendritic cell neoplasm (BPDCN). This is a rare subtype of acute leukaemia characterized by the clonal proliferation of precursors of plasmacytoid dendritic cells, also known as professional type I interferon-producing cells or plasmacytoid monocytes.

In a preferred embodiment the leukaemia is a MLL-rearranged (MLLr) leukaemia.

The term "MLL-rearranged" or "MLLr" when referred to leukaemias, as used herein, refers to a leukaemia in which the MLLl gene is disrupted. The MLLl gene, also named mixed- lineage leukemia 1, is now renamed lysine [K] -specific methyltransferase 2A or KMT2A. The complete protein sequence for human MLLl has the UniProt accession number Q03164 (June 7, 2017). The MLLl gene is on chromosome l lq23 and in MLL-rearrangements is disrupted by different chromosomal rearrangements. These MLL-rearrangements include non-constitutional or acquired deletions, duplications, inversions and reciprocal translocations at l lq23. Translocations involving 1 lq23 result in MLLl gene fusioned in frame to more than 80 different partner genes. Alternatively, the MLL-rearranged leukaemia can be named "I lq23/MLL rearrangement". Exemplary I lq23/MLL rearrangements according to the present invention can be, without limitation, t(4;l 1), t(l 1 ; 19), t(9;l 1), t(l lq23;V). In an embodiment the rearrangement is selected from the group consisting of the t(4; 11)/MLL-AF4 (MA4) rearrangement, the t(10;l 1)/MLL-AF10 rearrangement, and the t (1;1 l)(p32;q23)/MLL-EPS15 rearrangement. In a preferred embodiment, the rearrangement is t(4; 11)/MLL-AF4 (MA4). In t(4; 11)/MLL-AF4 (MA4) the AF4 protein (ALL 1 -fused gene from chromosome 4) is fused in-frame to MLLl as a result of a t(4; I l)(q21,q23) translocation. In another preferred embodiment, the rearrangement is t(l;l l)(p32;q23)/MLL-EPS15. In t(l;l l)(p32;q23)/MLL-EPS15 the EPS 15 protein is fused in-frame to MLLl as a result of a t(l;l I)(p32;q23) translocation. In another embodiment, the rearrangement is t(l l;19) (q23;pl3). MLL rearrangements may be identified by techniques known by the person skilled in the art such as Southern blot, RT-PCR or genomic long-range PCR.

The term "MLLr leukaemia", in the context of the present invention, encompasses infant, pediatric and adult leukaemias. Therapy-related leukaemias having MLLl rearrangements, attributable to prior treatment with certain chemotherapeutic agents, particularly topoisomerase II inhibitors, are also encompassed by this term. In a preferred embodiment, the MLLr leukaemia is selected from the group consisting of MLLr AML and MLLr ALL.

In another embodiment, the MLLr leukaemia is an acute leukaemia. In a preferred embodiment, the leukaemia is MLLr B-cell acute leukaemia. In another embodiment, the MLLr B-cell acute leukaemia is selected from the group consisting of MLLr B-cell AML and MLLr B-cell ALL.

In a preferred embodiment, the leukaemia is MLLr B-cell acute lymphoblastic leukaemia, preferably MLLr infant B-cell acute lymphoblastic leukaemia (iMLLr-B- ALL).

The term "iMLLr-B-ALL" refers to an infant leukaemia carrying MLLr that has a distinctive pro-B/mixed phenotype (CD 10- with expression of myeloid markers) and frequently shows therapy refractoriness and central nervous system (CNS) infiltration.

In another embodiment, the leukaemia is a relapsed/refractory leukaemia. The expression "relapsed/refractory" or "R/R" refers to a relapsed leukaemia that has achieved a complete remission to initial treatment and then experience a recurrence, or to a refractory leukaemia that is resistant to a treatment by not achieving complete remission. Preferably, the R/R leukaemia is a R/R acute leukaemia, more preferably a R/R ALL, even more preferably a R/R B-ALL. In another embodiment, the R/R leukaemia is a MLLr leukaemia, preferably a MLLr ALL, more preferably a MLLr B- ALL, even more preferably a iMLLr-B-ALL. In another embodiment, the R/R leukaemia is t(4;l 1)/MLL-AF4, more preferably t(4;l 1)/MLL-AF4 B-ALL.

In a preferred embodiment the leukaemia is a NG2+ leukaemia. The expression "NG2+ leukaemia" refers to a leukaemia having leukaemic cells, preferably blasts, in which NG2 cell surface expression can be detected. Methods to detect expression of NG2 are well known in the art and are disclosed in the context of the second aspect of the invention. The methods and embodiments disclosed in the context of the second aspect of the invention are also applicable to the first aspect. In a preferred embodiment, the NG2 expression is detected by an anti-NG2 antibody, preferably the 7.1 anti-NG2 antibody. More preferably, said detection is carried out by flow cytometry.

A NG2+ leukaemia is a leukaemia having at least one leukaemic cell in which

NG2 cell surface expression can be detected. However, a NG2+ leukaemia may also have NG2- leukaemic cells. In a preferred embodiment, the NG2+ leukaemia of the invention has at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%), or 100%) of leukaemic cells having NG2 expression.

In a preferred embodiment the leukaemia is a leukaemia having increased levels of NG2-expressing cells with respect to a reference value. Methods to determine this increase in the expression are disclosed in the context of the second aspect of the invention. The methods and embodiments disclosed in the context of the second aspect of the invention are also applicable to the first aspect. The term "reference value", as used herein, relates to a predetermined criteria used as a reference for evaluating the values or data obtained from the samples collected from a subject. The reference value or reference level can be an absolute value; a relative value; a value that has an upper or a lower limit; a range of values; an average value; a median value; a mean value; or a value as compared to a particular control or baseline value. A reference value can be based on an individual sample value, such as for example, a value obtained from a sample from the subject being tested, but at an earlier point in time. The reference value can be based on a large number of samples, such as from population of subjects of the chronological age matched group, or based on a pool of samples including or excluding the sample to be tested. The reference value derives from a sample collection formed preferably by a mixture of the sample to be analyzed from normal individuals not affected by the disease. Said reference value can be determined by means of techniques well known in the state of the art, for example, determining the mean of the levels of NG2 protein measured in a sample taken from healthy subjects. In a preferred embodiment the reference value is obtained from a sample of healthy subjects. The reference value can also be obtained from the constitutively expressed proteins taken from the same subject to be analyzed. The reference value should be obtained from the same tissue or fluid as the sample analyzed.

In a preferred embodiment, the treatment is administered to a subject having NG2+ leukemic cells or having increased levels of NG2-expressing cells with respect to a reference sample. The reference sample can be a sample from a healthy subject or a sample from a subject suffering from leukaemia and not having NG2-expressing cells. The inventors have also found that the administration of NG2 inhibitors reduces the tumoral mass in bone marrow and facilitates the migration or movilization of the blasts from bone marrow to peripheral blood, thus preventing relapse of leukaemia. Therefore, in another embodiment, the NG2 inhibitor reduces the tumoral mass in bone marrow. In another embodiment, the NG2 inhibitor causes the migration of blasts from bone marrow to peripheral blood. In another embodiment, the NG2 inhibitor prevents the relapse of leukaemia.

Furthermore, the inventors have found that pre -treatment with NG2 inhibitors enhances the cytotoxicity of chemotherapy. Therefore, in another embodiment, the NG2 inhibitor enhances the cytotoxicity of chemotherapy or increases the sensitivity to chemotherapy.

When the blasts are in peripheral blood, they can be easily destroyed by conventional chemotherapy. Therefore, the NG2 inhibitors of the invention can be administered in combination with other therapies, particularly chemotherapy, more particularly cytotoxic therapies .

Therefore, in a preferred embodiment, the inhibitor is administered in combination with one or more therapeutic agents useful in the treatment of leukaemia, preferably useful in the treatment of MLLr B-cell acute leukaemia, even more preferably useful in MLLr-B-ALL, even more preferably useful in the treatment of iMLLr-B-ALL. Exemplary agents that can be used in combination with the NG2 inhibitor of the invention for the treatment of leukaemia can be, without limitation, L- asparaginase, dexamethasone, vincristine, prednisone, prednisolone, cytarabine, daunorubicin, methotrexate, cyclophosphamide, mercaptopurine, etoposide, mitoxantrone, thioguanine, teniposide, daunomycin, daunorubicin, doxorubicin, idarubicin, fludarabine, cladribine, topotecan, hydroxyurea, azacitidine, decitabine, imatinib, busulfan, omacetaxine, amsacrine, FLT-3 kinase inhibitors, such as PKC412, AC220/quizartinib, midostaurin or CEP-701/lestaurtinib; proteasome inhibitors such as bortezomib; HDAC inhibitors such as valproic acid, romidepsin, or vorinostat; hypomethylating agents such as zebularine, 5-azacitidine, clofarabine, cladribine or decitabine; immunotherapy such as blinatumomab, or engineered T-cells (CAR-T); PD98059; tipifarnib; DotlL inhibitors such as pinometostat or SYC-522; bromodomain inhibitors such as JQ1; LSD1 inhibitors such as tranylcypromine or GSK2879552; polycomb protein inhibitors such as DZNep and UNCI 999; obatoclax; venetoclax; CDK6 inhibitors such as palbociclib; CDK9 inhibitors such as flavopiridol or dinaciclib; and combinations thereof.

In a preferred embodiment, the inhibitor is administered in combination with one or more therapeutic agents useful in the treatment of MLLr-B-ALL selected from the group consisting of L-asparaginase, dexamethasone, vincristine, prednisone, prednisolone, cytarabine, daunorubicin, methotrexate, cyclophosphamide, mercaptopurine, etoposide, mitoxantrone, thioguanine and combinations thereof. In a more preferred embodiment, the NG2 inhibitor is administered in combination with L- asparaginase, dexamethasone and vincristine. In another preferred embodiment, the NG2 inhibitor is administered in combination with dexamethasone. In another embodiment, the NG2 inhibitor is administered in combination with L-asparraginase, vincristine and a glucocorticoid. In another embodiment, the NG2 inhibitor is administered in combination with L-asparraginase, vincristine, a glucocorticoid and an anthracycline, preferably in combination with L-asparraginase, dexamethasone, vincristine and an anthracycline.

The expression "in combination", as used herein, has to be understood that the NG2 inhibitor of the invention can be administered together or separately, simultaneously, concurrently or sequentially with a therapeutic agent useful in the treatment of leukaemia in any order, e.g. the administration of the NG2 inhibitor can be made first, followed by the administration of one or more therapeutic agent(s) useful in the treatment of the disease; or the administration of the NG2 inhibitor of the invention can be made last, preceded by the administration of one or more therapeutic agent(s) useful in the treatment of the disease; or the administration of the NG2 inhibitor of the invention can be made concomitantly with one or more therapeutic agent(s) useful in the treatment of the disease. Preferably, the NG2 inhibitor is administered prior to the administration of one or more therapeutic agent(s) useful in the treatment of the disease, preferably conventional chemotherapeutic agents.

A person skilled in the art understands that the medicament for combined administration of the NG2 inhibitor and an additional therapeutic agent useful in the treatment of leukaemia can be in the form of a single dosage form or in separate dosage forms. The inventors have also found that NG2+ leukemic cells are more resistant to chemotherapy. Therefore, the invention also relates to a method for increasing sensitivity to chemotherapy comprising administering an NG2 inhibitor to a subject in need thereof, preferably to a subject suffering leukemia.

Method for designing a customized therapy

NG2 is not expressed in normal hematopoietic cells but it is specifically expressed in several leukaemias, such as MLLr leukaemias, both ALL and AML.

The inventors of the present invention have found that malignant cells from a

MLLr leukaemia, particularly from MLLr B-cell ALL, express NG2 in a significantly higher quantity compared to healthy donors. Therefore, the detection of the expression of NG2 in blood or other tissue cells can be useful for designing a customized therapy for a subject.

In another aspect, the invention relates to an in vitro method for designing a customized therapy for a subject diagnosed with leukaemia which comprises:

b) comparing said levels with a reference value

Designing a customized therapy to a subject diagnosed with leukaemia is understood as deciding, based on expression of NG2, administering as appropriate a NG2 inhibitor.

The first step of the second aspect of the invention comprises determining the levels of NG2-expressing cells in a sample from said subject.

The expression "determining the levels of NG2-expressing cells", as used herein, refers to determining the level of expression of a biomarker (NG2) and/or the number of cells carrying this biomarker on its surface (i.e. a cell surface marker). Therein, the level of expression refers to the level of mRNA and/or the level of protein and/or the number of cells carrying a biomarker on its surface. Methods for detecting the expression can be based on detecting NG2 mRNA or protein, or they also can be based on determining the mRNA levels or protein levels and the levels of variants thereof, in a sample as a whole, in cells of a sample and/or in the non-cellular fraction of a sample.

Methods for detecting mRNA are well known in the art and include, e.g., realtime PCR (rtPCR), northern blotting, nanostring and microarray technologies.

By way of a non-limiting illustration, the expression levels are determined by means of the quantification of the levels of mRNA encoded by said genes. The latter can be quantified by means of using conventional methods, for example, methods comprising the amplification of mRNA and the quantification of the amplification product of said mRNA, such as electrophoresis and staining, or alternatively, by means of Northern blot and the use of suitable probes of the mRNA of the gene of interest or of its corresponding cDNA/cRNA, mapping with the SI nuclease, RT-PCR, hybridization, microarrays, etc. Similarly, the levels of the cDNA/cRNA corresponding to said mRNA encoded by the marker gene can also be quantified by means of using conventional techniques; in this event, the method of the invention includes a step of synthesis of the corresponding cDNA by means of reverse transcription (RT) of the corresponding mRNA followed by the synthesis (RNA polymerase) and amplification of the cRNA complementary to said cDNA.

In order to normalize the values of mRNA expression among the different samples, it is possible to compare the expression levels of the mRNA of interest in the test samples with the expression of a control RNA. A "control RNA", as used herein, relates to RNA whose expression levels do not change or change only in limited amounts. Preferably, the control RNA is mRNA derived from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions. Preferred housekeeping genes for use in the present invention include 18-S ribosomal protein, β-2-microglobulin, ubiquitin, cyclophilin, GAPDH, PSMB4, tubulin and β-actin.

Alternatively, it is also possible to determine the expression levels of NG2 gene by means of the determination of the expression levels of the proteins encoded by said gene, since if the expression of gene is increased, an increase of the amount of corresponding protein should occur and if the expression of gene is decreased, a decrease of the amount of corresponding protein should occur.

Virtually any conventional method can be used within the frame of the invention to detect and quantify the levels of proteins. By way of a non-limiting illustration, the expression levels are determined by means of antibodies with the capacity for binding specifically to the protein to be determined (or to fragments thereof containing the antigenic determinants) and subsequent quantification of the resulting antigen-antibody complexes. The antibodies that are going to be used in this type of assay can be, for example, polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, scFv, diabodies, triabodies, tetrabodies and humanized antibodies. At the same time, the antibodies may or may not be labeled. Illustrative, but non-exclusive, examples of markers that can be used include radioactive isotopes, enzymes, fluorophores, chemoluminescent reagents, enzyme cofactors or substrates, enzyme inhibitors, particles, dyes, etc. There is a wide variety of well-known assays that can be used in the present invention, using non-labeled antibodies (primary antibody), labeled antibodies (secondary antibodies) or labeled antagonists or agonists of NG2; these techniques include Western-blot or immunoblot, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, immunofluorescence, techniques based on the use of biochips or protein microarrays including specific antibodies or assays based on the colloidal precipitation in formats such as reagent strips. Other forms of detecting and quantifying the proteins include affinity chromatography techniques, ligand-binding assays, etc.

In an embodiment of the invention, the NG2-expressing cells are detected by

Western blot, immunocytochemistry or flow cytometry.

In a preferred embodiment of the invention, the NG2-expressing cells are detected by immunocytochemistry, preferably by immunofluorescence, more preferred by flow cytometry.

"Immunocytochemistry" refers to a technique used to localize the presence of a specific protein or antigen in cells by use of a specific primary antibody that binds to it wherein the extracellular matrix and other stromal components are removed, leaving only whole cells to stain.

In a preferred embodiment of the invention, detecting the expression or determining the levels of NG2 is performed by immunofluorescence. Immunofluorescence (IF) is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualisation of the distribution of the target molecule through the sample. IF is a widely used example of immunostaining and is a specific example of immunohisto-chemistry (IHC) or immunocytochemistry (ICC) that makes use of fluorophores to visualise the location of the antibodies. IF can be used on tissue sections, cultured cell lines, or individual cells. IF can be used in combination with other, non-antibody methods of fluorescent staining, for example, use of DAPI to label DNA. Several microscope designs can be used for analysis of IF samples; the simplest is the epifluorescence microscope, and the confocal microscope is also widely used. Various super-resolution microscope designs that are capable of much higher resolution can also be used. In a preferred embodiment, the identification of a malignant cell is performed by flow cytometry, which is a laser- based, biophysical technology employed in cell counting, cell sorting and biomarker detection by suspending cells in a stream of fluid and passing them by an electronic detector.

As a person skilled in the art can know, the expression of NG2 can also be detected by detecting the expression of a functionally equivalent variant of said glycoprotein.

"Functionally equivalent variant" is understood to mean all those proteins derived from NG2 sequence by modification, insertion and/or deletion or one or more amino acids, whenever the function is substantially maintained.

Preferably, variants of NG2 are (i) polypeptides in which one or more amino acid residues are substituted by a preserved or non-preserved amino acid residue (preferably a preserved amino acid residue) and such substituted amino acid may be coded or not by the genetic code, (ii) polypeptides in which there is one or more modified amino acid residues, for example, residues modified by substituent bonding, (iii) polypeptides resulting from alternative processing of a similar mR A, (iv) polypeptide fragments and/or (v) polypeptides resulting from NG2 fusion or the polypeptide defined in (i) to (iii) with another polypeptide, such as a secretory leader sequence or a sequence being used for purification (for example, His tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated through proteolytic cut (including multisite proteolysis) of an original sequence. The variants may be post-translationally or chemically modified. Such variants are supposed to be apparent to those skilled in the art.

As known in the art, the "similarity" between two polypeptides is determined by comparing the amino acid sequence and the substituted amino acids preserved from a polypeptide with the sequence of a second polypeptide. The variants are defined to include polypeptide sequences different from the original sequence, preferably different from the original sequence in less than 40% of residues per segment concerned, more preferably different from the original sequence in less than 25% of residues per segment concerned, more preferably different from the original sequence in less than 10% of residues per segment concerned, more preferably different from the original sequence in only a few residues per segment concerned and, at the same time, sufficiently homologous to the original sequence to preserve functionality of the original sequence. The present invention includes amino acid sequences which are at least 60%>, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two polypeptides may be determined using computer algorithms and methods which are widely known to those skilled in the art. The identity between two amino acid sequences is preferentially determined using BLASTP algorithm [BLASTManual, Altschul, S. et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990)].

"Functionally equivalent variants" also include post-translational modifications of NG2 protein whenever the function is substantially maintained.

The term "sample", as used herein, refers to any sample containing tumoral cells. In a preferred embodiment, the sample is tumor tissue. Exemplary tumor tissue that can be used in the present invention are, without limitation, skin, soft tissue, bone, pleura, testicles, visceral organs such as kidney, liver, or spleen, etc. Said sample can be obtained by conventional methods, e.g. biopsy, surgical excision or aspiration, by using methods well known to those of ordinary skill in the art, such as gross apportioning of a mass, or microdissection or fine needle aspiration cytology. In another embodiment, the sample is a biofluid. In a preferred embodiment the biofluid is selected from the group consisting of blood and cerebrospinal fluid. In another embodiment the sample are cells, preferably blast cells.

In a more preferred embodiment the expression level of NG2 is determined in a sample selected from the group consisting of bone marrow, blood, cerebrospinal fluid and lymph nodes; more preferably selected from bone marrow and blood.

A sample from a bone marrow can be obtained by aspiration and trephine biopsy as known in the art. Blood samples can be obtained by conventional methods, using processes known in the state of the art by the person skilled in the art, such as blood extraction by means of puncturing an artery or vein, normally a vein from the inner part of the elbow or from the back of the hand, the blood sample being collected in an airtight vial or syringe. Cerebrospinal fluid can be obtained by lumbar puncture. Lymph nodes are obtained by biopsy of all or part of a lymph node (excisional lymph node biopsy or incisional lymph node biopsy).

In a preferred embodiment the blood sample is peripheral blood.

"Peripheral blood", as used herein, refers to a sample comprising the cellular components of blood, consisting of red blood cells, white blood cells, and platelets, which are found within the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver, or bone marrow.

The sample may be assayed as a whole sample, e.g. in crude form. Alternatively, the sample may be fractionated prior to analysis, e.g. by density gradient centrifugation, fluorescence activated cell sorting, etc. to purify leukocytes or one or more fractions thereof, e.g. blast cells.

In a preferred embodiment, the NG2-expressing cells are hematopoietic cells, preferably blasts. Hematopoietic cells and blasts can be isolated from samples by methods well known by the skilled in the art, such as those described in the examples of the present patent application. By way of illustration, blasts can be isolated by using antibodies against CD45, CD19, CD10, CD34 and NG2, for example by FACS- immunophenotyped using the monoclonal antibodies CD45-FITC, CD19-APC, CD 10- PerCP-Cy5.5, CD34-PE-Cy7 (BD Biosciences) and NG2-PE (Beckman). Alternatively, cells can be stained with antibodies against HLA-ABC and CD45 to identify human leukemia by flow cytometry and then immunophenotyped using antibodies against CD 19, CD 10, CD34, CD33 and NG2 antigens. Methods for isolating blast populations are described in Stam RW. et al. Blood 2010, 115(14): 2835-2844.

The term "subject" or "individual" or "animal" or "patient" includes any subject, particularly a mammalian subject, for whom therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

In a second step the in vitro method of the second aspect of the invention comprises comparing the level of NG2 with a reference value. Said comparison allows concluding if the subject is to be treated with a NG2 inhibitor.

The term "reference value", as used herein, relates to a predetermined criteria used as a reference for evaluating the values or data obtained from the samples collected from a subject. The reference value or reference level can be an absolute value; a relative value; a value that has an upper or a lower limit; a range of values; an average value; a median value; a mean value; or a value as compared to a particular control or baseline value. A reference value can be based on an individual sample value, such as for example, a value obtained from a sample from the subject being tested, but at an earlier point in time. The reference value can be based on a large number of samples, such as from population of subjects of the chronological age matched group, or based on a pool of samples including or excluding the sample to be tested.

The reference value derives from a sample collection formed preferably by a mixture of the sample to be analyzed from normal individuals not affected by the disease. The reference value should be obtained from the same tissue or fluid as the sample analyzed. Said reference value can be determined by means of techniques well known in the state of the art, for example, determining the mean of the levels of NG2 protein measured in a sample taken from healthy subjects. In a preferred embodiment, the reference value is obtained from healthy subjects. The reference value can also be obtained from the constitutively expressed proteins taken from the same subject to be analyzed.

The term "increase of the expression level" is referred to the level of expression of NG2 which is higher than a reference value. The levels of expression are considered to be higher than its reference value when they are at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more higher than its reference value.

The term "decrease of the expression level" refers to the level of expression of NG2 which is lower than a reference value. The expression level is considered to be lower than a reference value when it is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more lower than its reference value.

In an embodiment the leukaemia is MLLr B-cell acute lymphoblastic leukaemia. In another embodiment the rearrangement is selected from the group consisting of the t(4;l l)/MLL-AF4 (MA4) rearrangement and the t(l;l l)(p32;q23)/MLL-EPS15 rearrangement. In another embodiment the inhibitor is selected from the group consisting of chondroitinase, an antibody, interference RNA, an antisense oligonucleotide, a ribozyme, an aptamer and an spiegelmer; preferably an antibody and chondroitinase. In an embodiment the expression level of NG2 is determined by measuring the level of mRNA encoded by NG2 gene, or by measuring the level of NG2 protein or of variants thereof. In an embodiment the mRNA expression level is determined by PCR or the expression level of proteins or of variants thereof are determined by flow cytometry.

All the terms and embodiments previously described in the context of the first aspect of the invention are equally applicable to this aspect of the invention.

Method for determining tumor resistance or sensitivity to a chemotherapeutic agent

The inventors have also found that NG2+ leukemic cells are resistant to chemotherapeutic agents, particularly to a treatment with dexamethasone or to a treatment with a combination of dexamethasone, L-asparaginase and vincristine. Thus, in a third aspect, the invention relates to an in vitro method for determining whether a tumor is resistant or sensitive to chemotherapy in a subject suffering from leukaemia comprising:

b) comparing said levels with a reference value,

The terms "subject", "leukaemia" "NG2 -expressing cells", "sample" have been defined previously and are also applicable to this aspect of the invention.

In the context of the third method of the invention the "tumor" is a leukaemic tumor.

Thus, in a first step of the method of the invention, the levels of NG2-expressing cells are determined in a sample from the patient. Methods for determining the levels of NG2-expressing cells in a sample from the patient have been disclosed in the context of the second aspect of the invention and are also applicable to the third aspect.

In a second step of the method of the invention, the levels of NG2 -expressing cells are compared to a reference value.

In the context of the method of the invention for determining tumor resistance to a chemotherapeutic agent, the "reference value" is the level of NG2-expressing cells determined in a sample from a healthy subject, a subject not suffering from cancer, or a tumor sample from a subject suffering from leukemia wherein said tumor is sensitive to chemotherapy. Preferably, the reference value is the level of NG2- expressing cells determined in a tumor sample from a subject suffering from leukemia wherein said tumor is sensitive to chemotherapy, more preferably wherein said tumor is sensitive to dexamethasone or to a combination of dexamethasone, L-asparaginase and vincristine.

In the context of the method of the invention for determining tumor sensitivity to a chemotherapeutic agent, the reference value is the level of NG2- expressing cells determined in a tumor sample from a subject suffering from leukemia wherein said tumor is resistant to chemotherapy.

Once this reference value is established, the level of NG2-expressing cells in the sample can be compared with this reference value, and thus be assigned a level of "increased" or "decreased" expression. The terms "increased" and "decreased" have been defined in the context of the second aspect of the invention and are also applicable to the third aspect.

Thus, increased levels of NG2-expressing cells when compared to a reference value are indicative that the tumor is resistant to chemotherapy. Alternatively, decreased levels of NG2-expressing cells wen compared to a reference value are indicative that the tumor is sensitive to chemotherapy.

The expression "resistant to chemotherapy", as used herein, refers to the fact that a number of leukemic cells survive chemotherapy. Preferably, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%) of cells survive chemotherapy.

The expression "sensitive to chemotherapy", as used herein, refers to the fact that a number of leukemic cells are killed by chemotherapy. Preferably, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%), at least 99% or 100% of cells are killed by chemotherapy.

The skilled person can easily assess whether a cancer is a resistant cancer by assessing cell viability after treatment with chemotherapy as disclosed in example 8.

Chemotherapy according to the invention has been defined previously in the context of the second aspect of the invention. In a particular embodiment, the chemotherapy is standard chemotherapy, more particularly is an agent selected from the group consisting of dexamethasone, L-asparaginase, vincristine and combinations thereof. In an embodiment is dexamethasone. In another embodiment is a combination of L-asparaginase, vincristine and dexamethasone.

In an embodiment the leukaemia is MLLr B-cell acute lymphoblastic leukaemia. In another embodiment the rearrangement is selected from the group consisting of the t(4; l l)/MLL-AF4 (MA4) rearrangement and the t(l ;l l)(p32;q23)/MLL-EPS 15 rearrangement. In another embodiment the sample is peripheral blood. In an embodiment the expression level of NG2 is determined by measuring the level of mR A encoded by NG2 gene, or by measuring the level of NG2 protein or of variants thereof. In an embodiment the mRNA expression level is determined by PCR or the expression level of proteins or of variants thereof are determined by flow cytometry. All the terms and embodiments previously described are equally applicable to this aspect of the invention.

Combination of the invention The inventors have shown that NG2 inhibitors synergize with VxL-based induction therapy resulting in an extensive mobilization of iMLLr-B-ALL blasts from bone marrow into peripheral blood where they become more accessible/sensitive to conventional VxL-based chemotherapy resulting in higher complete remission rates (CRR) and consequently higher EFS and delayed relapse.

Therefore, in another aspect, the invention relates to a combination comprising a neuron-glial antigen 2 (NG2) inhibitor and one or more therapeutic agents useful in the treatment of leukaemia, preferably a chemotherapeutic agent.

The term "combination", as used herein, refers to a material combination that comprises at least two components, as well as any product resulting, directly or indirectly, from the combination of the different components in any quantity thereof. This means that the NG2 inhibitor of the invention can be administered together or separately, simultaneously, concurrently or sequentially with a therapeutic agent useful in the treatment of leukaemia in any order, e.g. the administration of the NG2 inhibitor can be made first, followed by the administration of one or more therapeutic agent(s) useful in the treatment of the disease; or the administration of the NG2 inhibitor of the invention can be made last, preceded by the administration of one or more therapeutic agent(s) useful in the treatment of the disease; or the administration of the NG2 inhibitor of the invention can be made concomitantly with one or more therapeutic agent(s) useful in the treatment of the disease. In an embodiment the NG2 inhibitor is administered prior to the administration of one or more therapeutic agent(s) useful in the treatment of the disease, preferably conventional chemotherapeutic agents. In a preferred embodiment, the NG2 inhibitor is co -administered with the one or more therapeutic agent(s) useful in the treatment of the disease.

A person skilled in the art understands that the components of the combination can be included in the same or in separate formulations, i.e., the medicament for combined administration of the NG2 inhibitor and an additional therapeutic agent useful in the treatment of leukaemia can be in the form of a single dosage form or in separate dosage forms. The different therapeutic agents useful in the treatment of leukaemia can also be in the same or in separate formulations. The formulations may be combined for joint use as a combined preparation. The combination may be a kit-of-parts wherein each of the components is individually formulated and packaged.

The term "NG2 inhibitor" has been defined in the context of the first aspect of the invention. All the embodiments disclosed for the first aspect of the invention are also applicable to the combination of the invention.

In a preferred embodiment, the NG2 inhibitor is selected from the group consisting of chondrotinase, an antibody, interference R A, an antisense oligonucleotide, a ribozyme, an aptamer and an spiegelmer.

In a preferred embodiment, the inhibitor is a proteoglycan-degrading enzyme, preferably chondroitinase. In another embodiment, the inhibitor is an antibody capable of binding to NG2.

In an embodiment, the combination of the invention comprises a proteoglycan- degrading enzyme and a therapeutic agent useful in the treatment of leukaemia selected from the group consisting of dexamethasone, L-asparaginase, vincristine and combinations thereof, preferably comprises chondroitinase and dexamethasone, L- asparaginase and vincristine.

In another embodiment, the combination of the invention comprises an antibody capable of binding to NG2 and a therapeutic agent useful in the treatment of leukaemia selected from the group consisting of dexamethasone, L-asparaginase, vincristine and combinations thereof, preferably comprises an antibody capable of binding to NG2 and dexamethasone, L-asparaginase and vincristine. In a more preferred embodiment, the combination comprises the clone 7.1 anti-NG2 monoclonal antibody or an antibody binding to the same epitope than clone 7.1 anti-NG2 monoclonal antibody and a therapeutic agent useful in the treatment of leukemia selected from the group consisting of dexamethasone, L-asparaginase, vincristine and combinations thereof, preferably dexamethasone, L-asparaginase and vincristine.

The expression "therapeutic agent useful in the treatment of leukaemia" refers to an agent that is administered for treating leukaemia, particularly a chemotherapeutic agent. The term "chemotherapeutic agent" includes standard chemotherapy drugs, which generally attack any quickly dividing cell, targeted therapy agents and immunomodulatory agents.

Preferably, the agents useful in the treatment of leukaemia are useful in the treatment of MLLr B-cell acute leukaemia, even more preferably useful in MLLr-B- ALL, even more preferably useful in the treatment of iMLLr-B -ALL. Exemplary agents that can be used in combination with the NG2 inhibitor of the invention for the treatment of leukaemia can be, without limitation, L-asparaginase, dexamethasone, vincristine, prednisone, prednisolone, cytarabine, daunorubicin, methotrexate, cyclophosphamide, mercaptopurine, etoposide, mitoxantrone, thioguanine, teniposide, daunomycin, daunorubicin, doxorubicin, idarubicin, fludarabine, cladribine, topotecan, hydroxyurea, azacitidine, decitabine, imatinib, busulfan, omacetaxine, amsacrine, FLT- 3 kinase inhibitors, such as PKC412, AC220/quizartinib, midostaurin or CEP- 701/lestaurtinib; proteasome inhibitors such as bortezomib; HDAC inhibitors such as valproic acid, romidepsin, or vorinostat; hypomethylating agents such as zebularine, 5- azacitidine, clofarabine, cladribine or decitabine; immunotherapy such as blinatumomab, or engineered T-cells (CAR-T); PD98059; tipifarnib; DotlL inhibitors such as pinometostat or SYC-522; bromodomain inhibitors such as JQ1; LSD1 inhibitors such as tranylcypromine or GSK2879552; polycomb protein inhibitors such as DZNep and UNCI 999; obatoclax; venetoclax; CDK6 inhibitors such as palbociclib; CDK9 inhibitors such as flavopiridol or dinaciclib; and combinations thereof.

In an embodiment, the inhibitor is administered in combination with one or more therapeutic agents useful in the treatment of MLLr-B-ALL selected from the group consisting of a vinca alkaloid, a glucocorticoid, a cytotoxic enzyme, an antimetabolite, an anthracycline, an alkylating agent, a topoisomerase II inhibitor and combinations thereof. In a preferred embodiment, the therapeutic agent is selected from the group consisting of a cytotoxic enzyme, a glucocorticoid, a vinca alkaloid and combinations thereof; preferably the inhibitor is administered in combination with a cytotoxic enzyme, a glucocorticoid, and a vinca alkaloid. In a more preferred embodiment, the inhibitor is administered in combination with a cytotoxic enzyme, a glucocorticoid, a vinca alkaloid and an anthracycline.

In a preferred embodiment, the inhibitor is administered in combination with one or more therapeutic agents useful in the treatment of MLLr-B-ALL selected from the group consisting of L-asparaginase, dexamethasone, vincristine, prednisone, prednisolone, cytarabine, daunorubicin, methotrexate, cyclophosphamide, mercaptopurine, etoposide, mitoxantrone, thioguanine, and combinations thereof.

In a more preferred embodiment, the NG2 inhibitor is administered in combination with L-asparraginase, dexamethasone and vincristine. In another preferred embodiment, the NG2 inhibitor is administered in combination with dexamethasone or a pharmaceutically acceptable salt thereof.

In an embodiment, the therapeutic agent useful in the treatment of leukaemia is selected from the group consisting of vincristine, a glucocorticoid, L-asparaginase, anthracycline, and combinations thereof. In a preferred embodiment, the therapeutic agents are a combination of vincristine, a glucocorticoid and L-asparaginase. In another preferred embodiment, the therapeutic agents are a combination of vincristine, a glucocorticoid, L-asparaginase and an anthracycline, preferably L-asparaginase, dexamethasone, vincristine and an anthracycline.

In an embodiment of the combination of the invention the NG2 inhibitor is selected from the group consisting of chondroitinase, an antibody, interference R A, an antisense oligonucleotide, a ribozyme, an aptamer and an spiegelmer; and wherein the therapeutic agent useful in the treatment of leukaemia is selected from the group consisting of L-asparaginase, dexamethasone, vincristine, prednisone, prednisolone, cytarabine, daunorubicin, methotrexate, cyclophosphamide, mercaptopurine, etoposide, mitoxantrone, thioguanine, teniposide, daunomycin, daunorubicin, doxorubicin, idarubicin, fludarabine, cladribine, topotecan, hydroxyurea, azacitidine, decitabine, imatinib, busulfan, omacetaxine, amsacrine, FLT-3 kinase inhibitors, proteasome inhibitors, HDAC inhibitors, hypomethylating agents, immunotherapy, PD98059, tipifarnib, DotlL inhibitors, bromodomain inhibitors, LSDl inhibitors, polycomb protein inhibitors, obatoclax, venetoclax, CDK6 inhibitors, CDK9 inhibitors and combinations thereof. In an embodiment of the combination of the invention, the NG2 inhibitor is selected from the group consisting of chondroitinase, an antibody, interference R A, an antisense oligonucleotide, a ribozyme, an aptamer and an spiegelmer; and wherein the therapeutic agent useful in the treatment of leukaemia is selected from the group consisting of L-asparaginase, dexamethasone, vincristine, prednisone, prednisolone, cytarabine, daunorubicin, methotrexate, cyclophosphamide, mercaptopurine, etoposide, mitoxantrone, thioguanine, teniposide, daunomycin, daunorubicin, doxorubicin, idarubicin, fludarabine, cladribine, topotecan, hydroxyurea, azacitidine, decitabine, imatinib, busulfan, omacetaxine, amsacrine, PKC412, AC220/quizartinib, midostaurin, CEP-701/lestaurtinib, bortezomib, valproic acid, romidepsin, vorinostat, zebularine, 5- azacitidine, clofarabine, cladribine, decitabine, blinatumomab, engineered T-cells (CAR-T), PD98059, tipifarnib, pinometostat, SYC-522, JQ1, tranylcypromine, GSK2879552, DZNep, UNCI 999, obatoclax, venetoclax, palbociclib, flavopiridol, dinaciclib and combinations thereof.

In the present invention, when referred to a particular compound is also intended to encompass the pharmaceutically acceptable salt of said compound.

The term "pharmaceutically-acceptable salt" embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, example of which are formic, acetic, adipic, butyric, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, ethanedisulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, camphoric, camphorsulfonic, digluconic, cyclopentanepropionic, dodecylsulfonic, glucoheptanoic, glycerophosphonic, heptanoic, hexanoic, 2-hydroxy-ethanesulfonic, nicotinic, 2-naphthalenesulfonic, oxalic, palmoic, pectinic, persulfuric, 2-phenylpropionic, picric, pivalic propionic, succinic, tartaric, thiocyanic, mesylic, undecanoic, stearic, algenic, [beta]-hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable pharmaceutically-acceptable base addition salts include metallic salts, such as salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or salts made from organic bases including primary, secondary and tertiary amines, substituted amines including cyclic amines, such as caffeine, arginine, diethylamine, N-ethyl piperidine, aistidine, glucamine, isopropylamine, lysine, morpholine, N-ethyl morpholine, piperazine, piperidine, triethylamine, trimethylamine. All of these salts may be prepared by conventional means from the corresponding compound of the invention by reacting, for example, the appropriate acid or base with the compound of the invention. When a basic group and an acid group are present in the same molecule, a compound of the invention may also form internal salts. The preparation of salts can be carried out by methods known in the art.

Each of the formulations includes a pharmaceutically acceptable carrier. The terms "pharmaceutically acceptable carrier", or "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent,", or "pharmaceutically acceptable vehicle," used interchangeably herein, refer to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. A pharmaceutically acceptable carrier is essentially non-toxic to recipients at the dosages and concentrations employed, and is compatible with other ingredients of the formulation. Suitable carriers include, but are not limited to water, dextrose, glycerol, saline, ethanol, and combinations thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents.

Each of the components of the combination may be administered by a different route or by the same route.

In a particular embodiment, any of the formulations of the components of the combination is a formulation for parenteral administration. Thus, said formulation suitable for parenteral injection, include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or may comprise sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous or non-aqueous excipients or carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, triglycerides, including vegetable oils such as olive oil, or injectable organic esters such as ethyl oleate. In a more particular embodiment, any of the formulations of the components of the combination of the invention is a formulation for intravenous, intraperitoneal, intramuscular or subcutaneous administration. Typically, formulations for intravenous, intraperitoneal, intramuscular or subcutaneous administration are solutions in sterile isotonic aqueous buffer. If necessary, the formulation also includes a local anesthetic to ameliorate any pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachet indicating the quantity of active ingredient. Where the formulation is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the formulation is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. In an even more particular embodiment, the formulation of the invention is a formulation for intravenous or intraperitoneal administration.

In another particular embodiment, any of the formulations of the components of the combination of the invention is a formulation for oral administration.

Solid dosage forms for oral administration include conventional capsules, sustained release capsules, conventional tablets, sustained-release tablets, chewable tablets, sublingual tablets, effervescent tablets, pills, suspensions, powders, granules and gels. In the solid dosage forms, the active ingredients are admixed with at least one suitable excipient or carrier, such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, such as for example, starches, lactose, sucrose, mannitol, or silicic acid; (b) binders, such as for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, or acacia; (c) humectants, such as for example, glycerol; (d) disintegrating agents, such as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, or sodium carbonate; (e) solution retarding agents, such as for example, paraffin; (f) absorption accelerators, such as for example, quaternary ammonium compounds; (g) wetting agents, such as for example, cetyl alcohol or glycerol monostearate; (h) adsorbents, such as for example, kaolin or bentonite; and/or (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules and tablets, the dosage forms may also comprise buffering agents. Solid formulations of a similar type may also be used as fillers in soft or hard filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols, and the like. Solid dosage forms such as coated tablets, capsules and granules can be prepared with coatings or shells, such as enteric coatings and others known in the art. They may also contain opacifying agents, and can be formulated such that they release the active ingredient or ingredients in a delayed manner. Examples of embedding formulations that can be used are polymeric substances and waxes. The active ingredients can also be in micro-encapsulated form, if appropriate, with one or more of the aforementioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing suitable excipients or carriers used in the art. In addition to the active ingredients, the liquid dosage form may contain one or more excipients or carriers commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, particular cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame seed oil, Miglyol^®, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, or mixtures of these substances, and the like. In addition to said inert diluents, the formulation can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfuming agents. Suspensions, in addition to the active ingredient or ingredients, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol or sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar, or tragacanth, or mixtures of these substances, and the like.

Sustainable-release forms and appropriate materials and methods for their preparation are described in the art. In a particular embodiment, the orally administrable form of the formulation is in a sustained release form that further comprises at least one coating or matrix. The coating or sustained release matrix include, without limitation, natural polymers, semisynthetic or synthetic water-insoluble, modified, waxes, fats, fatty alcohols, fatty acids, natural semisynthetic or synthetic plasticizers, or a combination of two or more of them. Enteric coatings may be applied using conventional processes known to experts in the art.

The appropriate dosage of the active principle or principles within the combination will depend on the type of NG2 inhibitor, the type of cancer to be treated, the severity and course of the disease, previous therapy, the patient's clinical history and response to the NG2 inhibitor, and the discretion of the attending physician. The amount of NG2 inhibitor is suitably administered to the patient at one time or over a series of treatments. Depending on the time and severity of the disease, an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day, more preferably about 0.1 to 50 mg/kg per day, even more preferably 10 mg/kg/day. In another embodiment, the dose of the NG2 inhibitor, particularly chondroitinase, can be between 0.01-300 U. The dose of the further therapeutic agents useful in the treatment of leukemia is the dose usually administered for the treatment of leukemia which is known to the person skilled in the art. The ratio between the components that are part of the compositions used in the combination of the invention is not critical and can be adjusted by the skilled person depending on the antitumor agent used in each particular well as the desired indication. Thus, the invention encompasses compositions wherein the ratio between the quantities of the NG2 inhibitor and the further therapeutic agent can range from 100: 1 to 1 : 100, preferably 50:1 to 1 :50, in particular from 20: 1 to 1 :20, more particularly from 1 : 10 to 10: 1, or even more particularly from 5: 1 to 1 :5.

Another aspect of the invention is the combination of the invention for use in medicine. Alternatively, the invention relates to the use of the combination of the invention for the manufacture of a medicament.

In another aspect, the invention relates to a method for treating leukaemia comprising administering the combination of the invention to a subject in need thereof.

In a particular embodiment, the administration of the NG2 inhibitor starts before the administration of the therapeutic agent useful in the treatment of leukaemia. I a particular embodiment, the administration of the NG2 inhibitor starts first and, after a period of time and once the administration of the NG2 inhibitor has finished, the administration of the therapeutic agent useful in the treatment of leukaemia starts. In an alternative particular embodiment, the administration of the NG2 inhibitor overlaps in time with the administration of the therapeutic agent useful in the treatment of leukaemia. In this particular embodiment, the administration of the NG2 inhibitor starts first and, after a period of time, the administration of the therapeutic agent useful in the treatment of leukaemia begins while the administration of the NG2 inhibitor goes on. In a preferred embodiment, all the components of the combination of the invention are co- administered or administered at the same time.

The embodiments disclosed in the context of the first, second and third aspects of the invention are also applicable to this aspect of the invention.

The invention will be described by way of the following examples which are to be considered as merely illustrative and not limitative of the scope of the invention.

EXAMPLES

MATERIAL AND METHODS FOR EXAMPLES 1-5

Patient samples and clinical data

Clinical data was available for 67 diagnostic iMLLr-B-ALLs. Fifty-five of these infants were enrolled in the Interfant treatment protocol (Pieters R. et al. Lancet 2007; 370(9583): 240-250). Clinico-biological correlations in Figure 1 are based on the Interfant cohort except CNS disease data, which was obtained from twelve iMLLr-B- ALL used for xenotransplantation studies. Table 1 shows the clinico-biological data of the patients contributing samples for the experiments. The Institutional Review Board of the Hospital Clinic of Barcelona approved the study, and all patients' parents gave written informed consent. Mononuclear cells (MNCs) from patients with >85% of blasts were isolated from diagnostic bone marrow (BM) or peripheral blood (PB) by density gradient centrifugation using Ficoll-Hypaque. Blasts were FACS-immunophenotyped using the monoclonal antibodies CD45-FITC, CD19-APC, CD10-PerCP-Cy5.5, CD34- PE-Cy7 (BDBiosciences) and NG2-PE (Beckman), and NG2+ and NG2- blast populations were FACS-sorted (FACS Aria) (Figure 2A).

01 n.d. F MLL-AF4 n.a. 185 Neg 38 CR Neg Neg n.d.

02 7 M MLL-AF4 WT 300 Pos 56 Exitus Pos Neg Pos

03 10 M MLL-AF10 WT 21 Neg 45 CR Pos Pos Pos

04 6 F MLL-EPS15 WT 96 Neg 47 CR Pos Pos n.d.

05 6 F MLL-AF4 n.a. 515 Neg 84 Exitus Pos Neg n.d.

06 1.4 F MLL-AF4 WT 1332 Pos 59 Exitus Pos Pos Pos

07 6.5 M MLL-AF4 WT 204 Pos 48 Exitus Pos Neg Pos

08 3.5 M MLL-AF4 Mut 349 Pos 68 Exitus Pos Pos n.d.

09 1.9 F MLL-AF4 WT 263 Pos 42 CR Pos Pos Pos

10 3 F MLL-AF4 Mut 499 Pos 54 CR Pos Neg n.d.

11 2 F MLL-AF4 Mut 57 n.a. 37 CR Neg Neg n.d.

12 6.5 F MLL-AF4 Mut 337 Pos 33 Exitus Pos Pos n.d.

6 CR-6

Mean 4.9 351 64% " . 83% 50% 100%

Exitus

Table 1: Clinico-biological features of patients included in this study. months; WT: wild -type/germline; Mut: Mutated; CR: complete remission; n.a.: non available; n.d.: not done

Mice transplantation and follow-up

Non-obese diabetic/LtSz-scid IL-2Ry^"/- mice (NSG; n=305) housed under pathogen-free conditions were used. All experimental procedures were approved by the Animal Care Committee of The Barcelona Biomedical Research Park (DAAM7393). Limiting dilution doses (200k, 50k, 20k, 10k, 5k and lk) of sorted NG2⁺ and NG2^" leukemic blasts were intra-bone marrow-transplanted (IBM-transplanted) into sublethally irradiated mice as previously described (Bueno C. et al. Cytotherapy 2010, 12(1): 45-49; Monies R. et al. Leukemia 2014, 28(3): 666-674; Romero-Moya D. et al. Haematologica 2013, 98(7): 1022-1029). PB was collected weekly to analyze leukemia engraftment by flow cytometry. Mice were sacrificed when i) disease symptoms were evident, ii) leukemia engraftment reached 10% in PB, or iii) 140 days after transplantation. For secondary transplantation, BM-derived MNCs were collected from primografts and IBM-transplanted (200k and 10k doses) into irradiated secondary recipients (n=47) as above. For intravenous (IV) transplantation (n=15), 200k and 50k NG2⁺ or NG2^" sorted blasts were transplanted via the lateral tail vein as described (Sanjuan-Pla A. et al. Stem cells and development 2016, 25(3): 259-65). IV- transplanted mice were sacrificed and analyzed when human chimerism was detectable in PB.

Analysis of leukemia engraftment and PB hematologic counts

BM from injected tibia (IT), contralateral tibia (CL), liver, spleen and PB were collected and analyzed at sacrifice. Cells were stained with HLA-ABC-FITC and CD45- APC-Cy7 antibodies to identify human leukemia by flow cytometry. Leukemia was immunophenotyped using CD19-V450, CD10-PerCP-Cy5.5, CD34-PE-Cy7, CD33- APC and NG2-PE antibodies. Absolute white blood cell counts (WBC) and differential counts were determined in PB (Monies R. et al. Blood 2011, 117(18): 4746-4758). Hepatosplenomegaly was analyzed as described (Prieto C. et al. Cancer Research 2016, 76(8): 2478-2489). The t(4;l l)/MA4 rearrangement was confirmed by dual-fusion FISH in both diagnostic NG2+ and NG2- cell populations and in engrafted leukemic cells as described (Bueno C. et al. Carcinogenesis 2009, 30(9): 1628-1637; Munoz- Lopez A. et al. Stem Cell Reports 2016, 7(4): 602-618) Analysis of CNS infiltration

Mice skulls were retrieved at sacrifice, fixed, decalcified, embedded in paraffin, and cut-stained with hematoxylin and eosin (H/E) as previously described (Prieto C. et al. Cancer Research 2016, 76(8): 2478-2489). Ten skull sections/mouse were analyzed and classified by the presence/absence of infiltrating blasts. Human chimerism in skull was assessed by immunohistochemistry using the Benchmark automated staining instrument and the human antibodies CD 19 and CD45 (Roche).

NG2 expression in CNS-ICs was analyzed by immunofluorescence in skull sections, using spleen as a positive control. Primary antibodies used were rabbit anti- NG2 and rat anti-endomucin (Millipore). Secondary antibodies used were donkey anti- rabbit and anti-rat (LifeTechnologies). Human NG2 expression was confirmed by qRT- PCR in BM and extramedullar tissues. RNA from BM, liver and spinal cord of engrafted mice was cDNA-converted and used for RT-PCR as previously described (Prieto C. et al. Cancer Research 2016, 76(8): 2478-2489) using the following primers: NG2: Fwd-5 '-CCTCTGGAAGAACAAAGGTCTC-3 ' (SEQ ID NO: 1), Rev-5'- GAACTGTGTGACCTGGAAGAG-3 ' (SEQ ID NO: 2); GAPDH: Fwd-5 '- GGGAAGCTTGTCATCAATGGA-3 ' (SEQ ID NO: 3), Rev-5'- CGCCCCACTTGATTTTGG-3 ' (SEQ ID NO: 4). PCR conditions were 95°C (20 seconds) followed by 40 cycles of 95 °C (1 second) and 60°C (20seconds).

Microarray gene expression profiling

CD34⁺CD19⁺CD10^"NG2⁺ and CD34⁺CD19⁺CD10^"NG2^" blast populations were FACS-purified from the BM of three iMLLr-B-ALL for global gene expression profiling (GEP) as described (Stam RW. et al. Blood 2010, 115(14): 2835-2844). Hierarchical clustering of genes was performed with the one -minus-correlation metric and the unweighted average distance. Gene functions and canonical pathways was analyzed using Ingenuity Pathway Analysis (IP A) software. Microarray data was deposited in the public Gene Expression Omnibus database, accession number GSE19475.

To confirm gene expression changes a qPCR array was used to analyze the expression of 84 genes involved in epithelial-to-mesenchymal transition (EMT)/migration pathways (QIAGEN). The authors of the invention specifically compared circulating NG2⁺ and NG2^" blasts recovered from primografts transplanted with NG2⁺ versus NG2^" cells. PCR Array was performed on a Stratagene-Mx3000P System following manufacturer's instructions. Raw data were analyzed using the SABiosciences web-based tool. Genes showing > 1.5 -fold-change expression were considered differentially expressed between both groups. IPA software was used to predict top regulated pathways/gene functions (Ayllon V. et al. Leukemia, 29(8):1741- 53). Statistical analysis

Data are expressed as mean±standard errors (s.e) of independent experiments unless otherwise specified. Statistical comparisons were performed using either paired or unpaired Student t test, as appropriate. Fisher's exact test was used to assess the association between clinical characteristics and NG2 expression (high versus low levels). Statistical significance was defined as p-value<0.05. EFS was calculated as the time from diagnosis to first failure (induction failure, relapse, death or second neoplasm). EFS curves of patients and xenografts were estimated according to Kaplan- Meier and compared with the log-rank test. The Cox model was used to estimate the impact of NG2 expression on the cause-specific hazard-of-relapse. Analyses were performed with SPSS Software. L-IC frequency was calculated using ELDA software (htt : ybioin wehi.cdu.au/ software/ elda. ) based on limiting dilution transplantation assays (Hu Y. et al. Journal of Immunological Methods 2009, 347(1-2): 70-78).

MATERIAL AND METHODS FOR EXAMPLES 6-7

Materials

The 7.1 anti-NG2 monoclonal antibody (MoAb) for in vivo experiments of treatment was generated from hybridoma cell line clone 7.1 supplied by Dr. I. Bernstein from the Fred Hutchinson Cancer Research Center. The 7.1 anti-NG2 monoclonal antibody for detection of expression of NG2 was supplied by Beckman Coulter.

Chase (chondroitinase ABC) from Proteus vulgaris was supplied by Sigma. Sample Preparation

Mononuclear cells (MNCs) from two patients with>85% of blasts were isolated from diagnostic bone marrow (BM) or peripheral blood (PB) by density gradient centrifugation using Ficoll-Hypaque. One of said patients carried a t(4; 11)/MLL-AF4 (MA4) rearrangement and the other patient carried a t(l;l l)(p32;q23)/MLL-EPS15 rearrangement. Blasts were FACS-immunophenotyped using the monoclonal antibodies CD45-FITC, CD19-APC, CD10-PerCP-Cy5.5, CD34-PE-Cy7 (BDBiosciences) and NG2-PE (Beckman), and NG2+ and NG2- blast populations were FACS-sorted (FACS Aria).

Mice transplantation, treatment and follow-up

Non-obese diabetic/LtSz-scid IL-2Ry-/- mice housed under pathogen-free conditions were used. All experimental procedures were approved by the Animal Care Committee of The Barcelona Biomedical Research Park (DAAM7393). Sorted NG2+ and NG2- leukemic blasts were either IBM-transplanted into sublethally irradiated mice. PB was collected weekly to analyze leukemia engraftment by flow cytometry. Once leukemia engraftment reached 10% in PB, mice were treated either with 7.1 anti- NG2 monoclonal antibody (MoAb) daily (lOmg/Kg) or chondroitinase (Ch^'ase) every other day (0.06 U/mouse) for 7 days and then sacrificed. For intravenous (IV) transplantation NG2- or NG2+ sorted blasts were transplanted via the lateral tail vein as described (Figure 6).

For some experiments NG2+ sorted blasts were pre -treated overnight with either 7.1 MoAb (0.7 mg/ml) or Ch^'ase (0.1 U/mL) before the injection. IV -transplanted mice were sacrificed and analyzed when human chimerism was detectable in PB.

Cytotoxicity assay

4xl0⁵ NG2-sorted cells per mL were cultured in 96-well plates in StemSpan media (Stem Cell Technologies) supplemented with stem cell factor (100 ng/niL), FLT3 ligand (100 ng/niL), IL-3 (lOng/mL), IL-7 (lOng/mL) (PeproTech) and ITS (IX) (Gibco). The drug concentration ranges for the assay were 0.05-50 μΜ for vincristine (V) (Selleckchem), 0.05-50 μΜ for dexamethasone (X) (Sigma-Aldrich, Spain) and 0.05-50 U/mL for L-asparaginase (L) (Kidrolase®). The same concentration ranges of each drug were used for the combo (VXL) treatment. 48 h after, cell viability was measured using the annexin-V apoptosis detection kit (BD Biosciences) according to the manufacturer's instructions by flow cytometry on a FACSCanto-II cytometer (BD Bioscience). FACSDiva software (BD Bioscience) was used for flow cytometry. Data were expressed as percentage of alive cells (7AAD and Annexin V-PE negative staining).

MATERIAL AND METHODS FOR EXAMPLES 9-10 Patient samples and immunophenotyping

Leukemic samples at presentation were used from five independent iMLLr-B- ALL patients with complete immunophenotypic and molecular/cytogenetic diagnosis. Patients mononuclear cells with >85% CD45^lowCD19⁺CD34⁺CD10^"NG2⁺ MLLr blasts were isolated by density gradient centrifugation using Ficoll-Hypaque. MLL (l lq23) status was confirmed in the diagnostic laboratory by FISH. Blasts were immunophenotyped using the monoclonal antibodies (MoAb) CD45-FITC, CD19-APC, CD10-PerCP-Cy5.5, CD34-PE-Cy7 (BD Biosciences, San Jose, CA) and NG2-PE (Beckman, Barcelona, Spain), and the NG2⁺ and NG2^~ blast populations were isolated by fluorescence-activated cell sorting (FACS) using a FACSAria cell sorter (BD Biosciences). The Institutional Review Board of the Hospital Clinic of Barcelona approved the study, and all patients' parents gave written informed consent.

Drugs

Vincristine (Selleckchem) and dexamethasone (Sigma-Aldrich) were reconstituted in DMSO. L-asparaginase (Kidrolase®, EUSA Pharma) and Chondroitinase ABC (Ch'ase, Sigma-Aldrich) were reconstituted in PBS as per supplier^'s guidelines. Drugs were stored in aliquots at -20°C. The clone 7.1 MoAb- producing hybridoma was kindly provided by Professor Irwin Bernstein, Fred Hutchinson Cancer Centre, Seattle, WA. Anti-NG2 7.1 MoAb was produced and purified using standard methods as previously detailed (Prieto C. et al. Leukemia 2018, 32(3): 633-644). All drugs were administrated by intraperitoneal (i.p.) injection. Patient-derived xenografts (PDX) models, in vivo treatment and analysis of leukemia engraftment

Eight-to-14-week-old NOD.Cg-Prkdcscidll2rgtmlWjl/SzJ mice (NSG; n=116) housed under pathogen free conditions were used. All experimental procedures with mice were approved by the Animal Care Committee of The Barcelona Biomedical Research Park (HRH-17-0045-P2). Between l-2xl0⁵ iMLLr-B-ALL cells were intravenously (lateral tail vein) transplanted into sublethally (2.25 Gy) irradiated mice as described (Sanjuan-Pla A. et al. Stem cells and development 2016; 25(3):259-265). Leukemia engraftment was monitored through weekly PB withdrawn, and the human graft was immunophenotyped by flow cytometry using HLA-ABC-FITC combined with the MoAb indicated above. When human graft in PB was >0.5%, mice were homogeneously divided into the following treatment groups: i) control, ii) VxL alone, iii) VxL plus Ch'ase, and, iv) VxL plus 7.1 MoAb. Treatment schedules were as follow: vincristine (V, 0.15mg/kg) once a week for 2 weeks; dexamethasone (x, 5mg/kg) and L- asparaginase (L, lOOOU/kg) daily during five days for 2 weeks. This standard induction treatment is known as VxL treatment. Ch'ase (0.06U/mouse) and 7.1 MoAb (lOmg/kg) were administered daily for 7 days (when given alone) or for 14 days (in combination with VxL). BM aspirates were always performed at the beginning and end of each treatment. Minimal residual disease (MRD) was assessed in the BM of each mouse at the completion of the 15-day treatment schedule. Complete remission (CR) was defined as presence of <1% leukemic cells in BM (0.1% in PB). Treatment was then stopped and mice were left untreated for 30 further days to follow up potential relapse by weekly analysis of engraftment in PB. Event-Free survival (EFS) comparing mice treated with VxL vs VxL plus NG2 blockers was analyzed with Kaplan-Meier curves from the end of the induction treatment (day 15) up to day 45 after. A leukemic engraftment in PB>0.5% was established to define a relapse event. Mice were sacrificed at the end of the experiment.

In vitro chemoresistance assay

Primary cells were maintained in a humidified atmosphere with 5% C02 at 37°C. MLLr-B-ALL primary samples cells were cultured in Stemspam medium (Stem Cell Technologies, Vancouver, Canada) supplemented with SCF, FLT3 ligand, IL3, IL-7 (all from PeproTech). Bone marrow-derived mesenchymal stromal cells (BM-MSC) were obtained, grown and characterized, lxl 0⁵ MLLr-B-ALL blasts were co-cultured in a 96- well plate with/without 2xl0⁴ irradiated BM-MSCs for either 30 min or 24 h, and then exposed to 0.5 or 50μΜ of VxL for 40h. Viability (apoptosis) of CD19⁺ B-ALL blasts was measured using 7-AAD on a FACSCanto-II cytometer using FACSDiva software (BD Biosciences).

Statistical analysis

Data are expressed as Mean±SEM of independent experiments unless otherwise specified. Statistical comparisons were analyzed using either paired or unpaired Student's t-test, as appropriate, using GraphPad Prism software (version 6.0, GraphPad; La Jolla, CA). For MRD studies, data is expressed as median (range) and significant differences were analyzed by the Median test. CR rates were statistically compared using the Pearson's chi-square test. EFS curves were compared with the log-rank test. Statistical analyses were performed using the SPSS software. Statistical significance was defined as p-value <0.05.

RESULTS Example 1: High levels of NG2 are associated with a more immature/aggressive phenotype in iMLLr-B-ALL

The clinical impact of NG2 expression was analyzed in an Interfant cohort (n=55) with the exception of CNS disease data that was analyzed in the cohort (n=12) of iMLLr-B-ALL (mainly MA4) available for xenotransplantation (Table 1). Percentages of NG2 expression in the blast population (5-90%) were explored as potential thresholds for the definition of high (NG2^high) versus low (NG2^low) levels of NG2 expression and the outcome in these subgroups evaluated in terms of risk-of-relapse. The cut-off of 40% NG2 expression was associated with the highest hazard ratio (HR) of relapse, i.e. 1.75, suggesting that patients with NG2 expression >40% (NG2^hlgh) had a 75% increase in the hazard-of-relapse as compared with patients with NG2 expression <40% (NG2^l0W) and was thus chosen as the cut-off for NG2 subgroups definition in further analyses (Figure 1A). Accordingly, NG2^hlgh infants showed a consistent trend towards a more aggressive phenotype (31%±9 vs 50%±10 5-year EFS±SE, p=0.1, Figure IB) than NG2^low patients, that was associated to a more immature (CD 10^" or byphenotypic) phenotype (82% versus 68%, p=0.2, Figure 1C, left panel), higher WBC (85% vs 55% of patients with >100xl0⁹ WBC/L, p=0.01, Figure 1C, middle panel), and more common CNS disease (80% vs 50% of patients with CNS disease, p=0.1, Figure 1 C, right panel).

Example 2: NG2 is a malleable marker that does not enrich for L-IC in iMLLr-B- ALL

Increasing evidence based on maturation-dependent antigens including CD34,

CD 10 and CD20 suggests that there is no stem cell hierarchy in pediatric B-ALL. NG2 is specifically expressed in MLLr leukemia but its function remains enigmatic. Here, the inventors addressed whether NG2 expression defines a blast population enriched in leukemia initiating propagating cells (L-IC) activity. The ability of highly purified (FACS purity>98%, Figure 2A) NG2+ and NG2- blasts to initiate leukemia was interrogated by IBM transplantation into NSG mice (n=243) following a limiting dilution (200k to lk blasts) transplantation approach summarized in Figure 2B. The majority (83%) of iMLLr samples were able to transfer the leukemia onto primografts (Tables 1, 2).

Table 2. Number of engrafted primografts according to cell dose and cell population transplanted.

As expected, EFS decreased with increasing doses of blasts transplanted (Figure 2C); however, with the exception of a slightly (p=0.06) more aggressive behavior of NG2- blasts transplanted at very high dose (200k), no differences were observed in either EFS or frequency of engrafted mice between NG2+ and NG2- populations injected at decreasing cell doses (Figure 2C, Table 2). The estimated frequency of L-IC was similar between NG2+ and NG2- populations (Figure 2D). Importantly, both NG2+ and NG2- engrafted leukemias remained MLLr and mirrored the original pro-B phenotype (CD45+CD19+CD10-) (Figure 2E), with engrafted mice presenting very similar BM and extramedullary hematopoietic sites infiltration (Figure 2F), splenomegaly, high WBC counts and a skewed granulocytic-to-lymphoid cell representation in PB (Figure 2G). Interestingly, NG2 expression was malleable as determined by the ability of both NG2+ and NG2- populations to re-establish in vivo the original leukemia immunophenotype with a continuum expression of NG2 (Figures 2A,E).

For serial transplantation experiments, 200k or 10k leukemic cells from primografts were transplanted into secondary mice, rendering a lower EFS (higher aggressiveness, Figure 3A, Table 3) than that observed in primary recipients; however, no differences in EFS were observed between mice transplanted with NG2+ or NG2- primografts (Figure 3A, top panels), suggesting an overall enrichment of L-ICs in secondary recipients irrespective of NG2 expression. Importantly, secondary leukemias from NG2+ and NG2- primografts retained a pro-B phenotype (Figure 3A, bottom panels), similar infiltration levels in BM and extramedullary hematopoietic sites (Figure 3B), splenomegaly, high WBC counts and skewed differentiation towards the lymphoid compartment in PB (Figure 3C). Of note, the continuum NG2 expression was reproduced in secondary mice (Figure 3A, bottom panels), demonstrating the malleability of NG2 expression.

Table 3. Number of secondary engrafted mice according to cell dose and cell population transplanted.

Example 3: NG2 is up-regulated in extramedullary hematopoietic tissues in iMLLr-B-ALL

Despite the IBM transplantation of a pure population of NG2+ or NG2- blasts, all leukemic cells engrafting primary and secondary mice showed a re-establishment of the NG2 phenotype, reproducing the continuum observed in the original leukemia (Figures 2E, 3 A, 4A). However, NG2 expression followed a tissue-specific pattern upon primary and secondary transplantation (Figure 4A). Whereas -40% of engrafted blasts expressed NG2 in BM, ~65%> of blasts were NG2+ in extramedullary hematopoietic tissues (p=0.01, Figure 4A). This was confirmed by qRT-PCR, showing 7-fold higher NG2 expression in extramedullary sites than in BM (Figure 4B, left panel). Because leukemia reconstitution requires proper homing to and seeding in the BM followed by exit of proliferating blasts to extramedullary organs, NG2+ and NG2- blasts were IV-transplanted and engraftment was analyzed 8 weeks later. IV- transplanted NG2+ blasts engrafted extramedullary tissues in 100% of mice (8% reconstitution levels) while NG2- blasts did so only in 13% of recipients with -0.6% reconstitution levels (Figure 4B, right panel). Importantly, analysis of leukocytosis in PB of iMLLr-B-ALL patients showed that NG2^high iMLLr-B-ALL infants displayed a striking 3 -fold higher number of circulating blasts than NG2^low patients (p=0.01 , Figure 4C). Together, these data suggest that NG2 is upregulated in response to systemic infiltration/migration, which is suggestive of a homeostatic adaptation of leukemic cells. Example 4: NG2 is not a prospective marker for CNS-IC but is upregulated in almost all MLLr blasts entering the CNS

The inventors addressed whether NG2 is involved in CNS infiltration by MLLr blasts. CNS infiltration is common in iMLLr-B-ALL and up to 75% of relapses occur within the CNS. As previously reported, leukemia infiltrates were consistently found in meninges/leptomeningeal space, but were rarely found within brain parenchyma (Figure 5A, top panels). The presence of infiltrating human B-lymphoid blasts observed by H/E staining was always confirmed by histopathology for CD45 and CD 19 (Figure 5A, bottom panels). 8/11 (73%) primary leukemias tested for CNS -infiltrating potential reproduced the patient phenotype (Figure 5B, top panel). Interestingly, 2/8 (25%>) patients engrafting CNS in mice showed no CNS involvement throughout disease evolution, indicating that CNS-engrafting capacity seems more prevalent than suggested by diagnostic cerebral-spinal fluid (CSF) cytospins. Although the frequency of mice displaying CNS disease was identical (50%>) between NG2+ and NG2- transplanted groups (Figure 5B, bottom panel), almost all human blasts reaching the CNS were consistently NG2+, irrespective of the NG2 phenotype of the population transplanted (results not shown). In line with this result, NG2 expression was 55-fold higher in CNS (spinal cord) than in BM (Figure 5C). These data indicate that while NG2 is not a marker to prospectively identify CNS-infiltrating ability, it is highly upregulated in MLLr blasts seeding the CNS.

Example 5: Global gene expression profiling reveals a migratory signature of NG2+ MLLr blasts

To identify patterns of gene expression that might provide a molecular explanation for the biology of NG2 expression in MLLr, the inventors performed whole-genome GEP on FACS-purified NG2+ and NG2- primary cells from t(4;l 1)/MA4+ pro-B ALL infants. A heatmap representation of hierarchical clustering of genes differentially expressed (20%> up- or down-regulated; p<0.05) between NG2+ and NG2- primary blasts was obtained. A total of only 281 genes (4%) were differentially expressed between NG2+ and NG2- primary t(4;l 1)+ blasts. Of these, 142 (50.5%o) were up-regulated and 139 (49.5%>) down-regulated in NG2+ cells, indicating little transcriptomic differences between both cell subsets. To get insight into the biological functions affected by differentially expressed genes, the inventors used IPA software to compare NG2+ and NG2- primary t(4;l 1)+ blasts. The inventors found that 8/12 (67%o) significant biological processes predicted to be activated in the NG2+ blasts were associated with "leukemic cell migration/movement", which is compatible with the functional upregulation of NG2 observed in MLLr blasts infiltrating extramedullary tissues and CNS.

To functionally support and validate patient global gene expression (GEP) data, the inventors performed qPCR to quantify the expression of a panel of genes associated with EMT/migration in NG2+ and NG2- blasts harvested from primografts. Differentially expressed genes were analyzed by IP A, and it was found that the majority (33/41, 81%) of the significant biological processes were associated with "cell migration/movement" and were predicted to be activated (z-score>2) in NG2+ circulating blasts, further confirming a migratory signature observed in NG2+ MLLr blasts infiltrating extramedullary hematopoietic tissues and CNS.

Example 6: The in vitro blocking of NG2 decreases the migratory capacity of iMLLr-B-ALL

The experiment of Figure 4B, right panel was repeated but engraftment was analyzed 4 weeks later instead of 8 weeks. It was confirmed that 4 weeks after the intravenous transplant 100% of the IV-transplanted mice with NG2+ show a clear engraftment of the leukemia in peripheral blood whereas only the 50% of the IV- transplanted mice with NG2- show leukemia in peripheral blood (Figure 7). Furthermore, the mean of engraftment in mice transplanted with NG2+ blasts was three times higher (12% vs 4%). These data suggest a migratory capacity of NG2+ blasts because in order to survive and proliferate these blasts need first migrate to bone marrow and from this location they colonize different peripheral hematopoietic organs such as peripheral blood.

NG2+ blasts were pretreated with three compounds: i) Chondroitinase, also named Chase, which is an enzyme that breaks proteoglycans; ii) a monoclonal antibody (clone 7.1) against NG2; and iii) a monoclonal antibody (clone 9.2.21, Abeam) against NG2. The blocking of NG2 after a short in vitro incubation of the cells blocks nearly completely the leukemic engraftment. The levels are even inferior to those achieved with NG2- cells (Figure 8). This blocking is showed by very low levels of blasts in peripheral blood due to the incapability of blocked NG2+ blasts of migrating to the bone marrow and nest there. Therefore, the in vitro blocking of NG2 prevents the engraftment and the capability of the leukemia to reside in the bone marrow. Blast expression before and after the treatment with Chase was analyzed by FACS (Figure 9)·

Example 7. The in vivo blocking of NG2 induces the mobilization of leukemic blasts from bone marrow to peripheral blood

Immunodeficient mice were intraperitoneally treated with chondroitinase or 7.1 MoAb after the leukemic engraftment had been established. The leukemic engraftment comes from a patient carrying a t(4; 11)/MLL-AF4 (MA4) rearrangement and from a patient carrying a t(l;l l)(p32;q23)/MLL-EPS15 rearrangement. NSG mice were i.v. transplanted with leukemic blasts. After 4-5 weeks with active leukemia in bone marrow and peripheral blood, mice were treated during 6-7 days with Chase or 7.1 MoAb as shown in Figure 6. After the treatment, mice were sacrificed and the leukemic engraftment in BM and PB was analyzed. It was shown that the intraperitoneal route is a right route of administration for Chase or 7.1 MoAb because after mice were sacrifized it is possible to confirm that leukemic blasts have turned into negative NG2 both in BM and PB (Figure 10). Chase and 7.1 MoAb block efficiently NG2 and inhibit its detection.

After 7 days of treatment the leukemic engraftment decreased 3-4 times in BM and in parallel increased 2-3 times in PB referred to the day when the treatment started, day 0 (Figure 11 A). This shows that during the treatment the engraftment is lost in BM and increases in PB due to a destabilization of the engraftment in BM after the inhibition of NG2. As a consequence of the NG2 inhibition, blasts lost their capability to anchor in BM and are found in PB (Figure 11B). Example 8. NG2+ blasts are resistant to chemotherapy and pre-treatment with NG2 inhibitors enhances the cytotoxicity of chemotherapy

In vitro, NG2+ cells are much more resistant to both dexamethasone (if used alone it does not kill NG2+ blasts) (Figure 12A) and the standard B-ALL chemotherapy, dexamethasone, L-asparaginase and vincristine (IC₅₀ is 1000 times lower for NG2- cells) (Figure 12B). This data is shown in Figure 12 for n = 2 patients.

Then, it was assessed in vivo whether NG2+ cells are more resistant to chemotherapy in the presence and absence of NG2 blocking. The percentage of NG2+ leukemic cells that remain in peripheral blood after treating mice with a combination of vincristine, L-asparaginase and dexamethasone (VxL) is increased when compared to mice that did not received VxL treatment (Figure 13). Very interestingly, pre -treatment with chondroitinase ABC enhances the cytotoxicity of chemotherapy.

Example 9. Blocking NG2 in vivo results in a robust mobilization of MLLr-B-ALL blasts into PB

To further confirm the biological role of NG2, 10⁵ NG2+ MLLr-B-ALL cells (n=4 patients) were i.v. transplanted and when PB engraftment was >0.5%, mice were daily i.p. treated with the NG2 inhibitor Ch'ase (0.06U/mouse) or anti-NG2 7.1 MoAb (lOmg/Kg/mouse) for 7 days. At completion of the 7-day treatment, the primografts treated either NG2 inhibitor displayed a significant reduction of leukemic engraftment in BM coupled to a massive leukemic infiltration in PB, as compared to vehicle-treated primografts (p<0.01; Fig 14A,B), suggesting that blocking NG2 in vivo mobilizes MLLr-B-ALL blasts to PB. Importantly, PDX models reproduced the immunophenotype of the de novo primary leukemia, and blasts recovered from primografts treated with NG2 blockers were mainly NG2- (Fig 14C), confirming a direct role of NG2 in the migration and mobilization of MLLr-B-ALL blasts.

Example 10. NG2 antagonists synergize with VxL therapy rendering higher CR rates and EFS in pre-clinical PDX models of MLLr-B-ALL

The inventors first tested in vitro whether BM-MSCs protect NG2+ MLLr B-ALL primary blasts from VxL, the standard-of-care treatment for B-ALL. NG2+ blasts pre- exposed to BM-MSC for 30min or 24h displayed ~20% and ~40%, respectively, increased resistance to VxL, demonstrating a BM stroma-mediated chemoprotection of MLLr B-ALL blasts to standard-of-care induction therapy (Fig 14D). Mobilization of leukemic cells from BM to PB is clinically desirable because circulating blasts become more accessible (and sensitive) to cytotoxic treatments, due in part, to their detachment from the chemoprotective BM niche. The inventors therefore tested whether mobilization of MLLr B-ALL blasts to PB by either the NG2 antagonist Ch'ase (Fig 15) or 7.1 MoAb (Fig 16) synergize with VxL treatment in robust preclinical PDX models of MLLr B-ALL. Engrafted PDXs were treated with vehicle, VxL alone, VxL plus Ch'ase, or VxL plus 7.1 MoAb, and CR (MDR<1% leukemic cells in BM (approx. <0.1% in PB) was assessed at completion of the treatment (day 15). Mice were then left untreated for 30 days and relapse and EFS were determined (Fig 15 A). In contrast to vehicle-treated mice, VxL treatment was highly efficient and engrafted PDXs treated with two cycles of VxL (14 days) showed a massive decrease in leukemia burden in both BM and PB (Fig 15B, 16A). Importantly, however, despite comparable engraftment levels (~24%) in BM at the time of treatment initiation (day 0) (Fig 15C, 16B), the levels of B-ALL engraftment in BM at the end of treatment (day 15) was >3- fold lower when either Ch'ase (0.8% vs 2.2%, Fig 15B,D) or 7.1 MoAb (1.3% vs 2.95%, Fig 16A,C) were co-administered with VxL. As a consequence, the rate of animals achieving CR was double when NG2 antagonists were co -administered with VxL: 63% vs 33% for Ch'ase (Fig 15D) and 46% vs 20% for 7.1 MoAb (Fig 16C). In order to further assess the potential clinical impact of such a lower levels of MRD/higher CR rates, treatment was removed and PDXs were followed up for a 30-day period. Importantly, 45-day EFS was higher in the group treated with either Ch'ase (65% vs 31%, Fig 15E) or 7.1 MoAb (55% vs 40%, Fig 16D) than the VxL alone. Accordingly, at the time of sacrifice, mice in the VxL alone group had 50% higher leukemic burden than those that received VxL combined with NG2 antagonists (Fig 15F). Collectively, NG2 blockage overrides BM stroma-mediated chemoprotection through PB mobilization of MLLr-B-ALL blasts, thus becoming more accessible to conventional chemotherapy. CONCLUSIONS

Clinical data from the Interfant cohort of iMLLr-B-ALL demonstrated that high NG2 expression associates with lower event-free survival, higher number of circulating blasts and more frequent CNS disease/relapse. Serial xenotransplantation of primary MLL-AF4+ leukemias indicated that NG2 is a malleable marker that does not enrich for L-IC or CNS-IC in iMLLr-B-ALL; nevertheless, its expression was highly upregulated in MA4⁺ blasts infiltrating extramedullar hematopoietic sites and CNS. Indeed, gene expression profiling of primary blasts and primografts revealed a migratory signature of NG2+ MLLr blasts. This study provides new insights on the biology of NG2 in iMLLr- B-ALL, supporting the development of NG2-directed therapies to reduce the risk of CNS disease/relapse and to provide safer CNS-directed therapies for iMLLr-B-ALL.

The in vivo treatment of NSG mice with MLL+/NG2+ acute leukemia induce the destabilization of the engraftment in BM and promotes the blast exit/migration to PB. These data support the design of therapies with NG2 inhibitors to reduce the tumoral mass in BM. Furthermore, the migration of the blasts to PB after treatment with NG2 inhibitors allows their subsequent removal with cytotoxic therapies that are more effective in the destruction of peripheral blasts. Therefore, NG2 inhibitors can be used to movilize blasts to PB as well as other compounds such as G-CSF or anti-CXCR4.

The data of the inventors also show that MLLr-B-ALL blasts mobilized to PB with NG2 inhibitors do in fact become sensitized to conventional chemotherapy (VxL) as demonstrated by lower levels of MRD and therefore higher rates of CR at treatment completion, resulting in higher EFS and delayed time-to-relapse.

Claims

1. A neuron-glial antigen 2 (NG2) inhibitor for use in the treatment of leukaemia.

2. The NG2 inhibitor for use according to claim 1 , wherein the leukaemia is MLL- rearranged (MLLr) B-cell acute lymphoblastic leukaemia.

3. The NG2 inhibitor for use according to any one of claims 1 or 2, wherein the leukaemia has an MLL rearrangement selected from the group consisting of the t(4; 11)/MLL-AF4 (MA4) rearrangement and the t(l;l l)(p32;q23)/MLL-EPS15 rearrangement.

4. The NG2 inhibitor for use according to any one of claims 1 to 3, wherein said inhibitor is selected from the group consisting of chondroitinase, an antibody, interference R A, an antisense oligonucleotide, a ribozyme, an aptamer and an spiegelmer.

5. The NG2 inhibitor for use according to claim 4, wherein said inhibitor is selected from the group consisting of an antibody and chondroitinase.

6. The NG2 inhibitor for use according to any one of claims 1 to 5, wherein said inhibitor is administered in combination with one or more therapeutic agents useful in the treatment of leukaemia.

7. An in vitro method for designing a customized therapy for a subject diagnosed with leukaemia which comprises:

b) comparing said levels with a reference value

8. The method according to claim 7, wherein said inhibitor is selected from the group consisting of chondroitinase, an antibody, interference RNA, an antisense oligonucleotide, a ribozyme, an aptamer and an spiegelmer.

9. The method according to claim 8, wherein said inhibitor is selected from the group consisting of chondroitinase and an antibody.

10. An in vitro method for determining whether a tumor is resistant or sensitive to chemotherapy in a subject suffering from leukaemia comprising:

(a) determining the levels of NG2-expressing cells in a sample from said subject, and

(b) comparing said levels with a reference value,

wherein increased levels of NG2-expressing cells with respect to the reference value are indicative that the tumor is resistant to chemotherapy, and decreased levels of NG2-expressing cells with respect to the reference value are indicative that the tumor is sensitive to chemotherapy.

11. The method according to any one of claims 7 to 10, wherein the leukaemia is MLLr B-cell acute lymphoblastic leukaemia.

12. The method according to any one of claims 7 to 11, wherein the leukaemia has a MLL rearrangement selected from the group consisting of the t(4; 11)/MLL-AF4 (MA4) rearrangement and the t(l;l l)(p32;q23)/MLL-EPS15 rearrangement.

13. The method according to any one of claims 7 to 12, wherein the sample is peripheral blood.

14. The method according to any one of claims 7 to 13, wherein the expression level of NG2 is determined by measuring the level of mRNA encoded by NG2 gene by PCR or by measuring the level of NG2 protein or of variants thereof by flow cytometry.

15. The method according to any one of claims 10 to 14, wherein the chemotherapy is an agent selected from the group consisting of dexamethasone, L-asparaginase, vincristine and combinations thereof.

16. A combination comprising a neuron-glial antigen 2 (NG2) inhibitor and one or more therapeutic agents useful in the treatment of leukaemia.

17. The combination according to claim 16 comprising a NG2 inhibitor, a cytotoxic enzyme, a glucocorticoid, and a vinca alkaloid.

18. The combination according to claim 17 comprising a NG2 inhibitor, vincristine, a glucocorticoid and L-asparaginase.

19. The combination according to claim 18, wherein the glucocorticoid is dexamethasone .

20. The combination according to any one of claims 16 to 19, further comprising an anthracycline.

21. The combination according to any one of claims 16 to 20, wherein the NG2 inhibitor is selected from the group consisting of an antibody and chondroitinase.

22. The combination according to any one of claims 16 to 21 for use in medicine.

23. The combination according to any one of claims 16 to 22 for use in the treatment of leukaemia.