FR3070047A1 - BIOMARKERS OF NEUROBLASTOMA - Google Patents

Abstract

The present invention relates to a method for in vitro molecular characterization of a neuroblastoma tumor, comprising the following steps: a) obtaining a sample of said tumor; b) Measurement in this sample of the expression level of one or more of the 19 genes encoding the proteins selected from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2, c) Comparison of the level of expression of said gene (s) measured in step (b) with a value determined reference for the or each of said genes. The present application also relates to a modulator of the expression or activity of a protein selected from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexine A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2 for its use in the treatment of neuroblastoma.

Description

NEUROBLASTOME BIOMARKERS

FIELD OF THE INVENTION

The present invention relates to the field of oncology, in particular the diagnosis and treatment of neuroblastoma.

STATE OF THE ART

Cancer is a disease of humans and animals that results in uncontrolled growth of endogenous cells. The term "cancer" is used to refer to the formation of malignant tumors and neoplasms (tumors or carcinomas). Generally, cancer growth involves two successive stages, first local tumor growth and second, cancer spread.

Local tumor growth consists of the appearance of a tumor clone, from a "transformed" cell, under the action of carcinogenic agents, initiators and promoters. Cell proliferation leads to the formation of the tumor.

Cancer spread from the original tumor can be by regional spread and / or by development of secondary tumors called metastases. This phenomenon is also referred to as the metastatic phase.

Cancers derived from neural crest cells are characterized by the appearance of solid tumors composed of cells that arise from the differentiation of neural crest cells during embryogenesis.

The neural crest designates, in the craniate embryo, a population of transient and multipotent cells generated from the most dorsal region of the neural tube. Neural crest cells migrate throughout the embryo during development and give rise to a wide variety of cell types in adults. The neural crest is the source of melanocytes (with the exception of the pigment cells of the eye), a large part of the bones and cartilage of the facial skeleton and neck, some endocrine cells, and gives rise to all glial cells and most of the neurons in the peripheral nervous system.

Tumors originating from neural crest cells are diverse in nature and include in particular neuroblastoma, melanomas, pheochromocytomas, paragangliomas, chemodectomas, neurogangliomas, PEC tumors (perivascular epithelioid cell), melanotic neuro-ectodermal tumors (MNTI), medullary carcinomas of the thyroid, gastroenteropancreatic neuroendocrine tumors, Ewing's sarcoma and similar tumors, malignant peripheral nerve sheath tumors (MPNSTs) (also called "malignant Schwannoma", " Neurofibrosarcoma ", and" Neurosarcoma "), neurofibromas, perineuriomas, neurothecomas, nerve sheath myxomas, certain types of medulloblastomas, and atypical teratoid and rabdoid tumors.

Among these tumors originating from cells derived from neural crests, neuroblastoma is a pediatric cancer responsible for more than 15% of infant mortality linked to cancer.

There are several clinical stages of neuroblastoma:

Stages 1, 2 and 3 are characterized by the presence of a more or less large tumor, but strictly localized in the abdomen, sometimes along the spine or at the level of the adrenal glands;

Stage 4 corresponds to the metastatic form, with a poor prognosis;

The 4S stage is characterized by the presence of generally significant hepatic and cutaneous metastases, but never of bone metastasis. This rather peculiar form regresses in the vast majority of cases, either spontaneously or following mild chemotherapy.

The molecular characterization of tumors from embryonic tissue is difficult because there is no healthy tissue corresponding to the tissue of the tumor: in fact, the neural crest cells disappeared during development, and no longer exist in an organism mammal after birth. Thus, there is no benchmark tissue with which to compare the expression rates of genes expressed differently in cancer.

Neuroblastomas are highly heterogeneous. Currently, the molecular characterization of neuroblastoma tumors is mainly based on the detection of the amplification of the N-myc gene. Indeed, the presence of multiple copies of the N-myc oncogene in a tumor cell is associated with a high risk of developing metastases, therefore for the tumor to progress towards metastatic stage 4.

Regarding the treatment of neuroblastoma, it is based on conventional techniques such as surgery, radiotherapy including brachytherapy, chemotherapy including hormone therapy, immunotherapy and more recently anti-angiogenic therapy.

However, despite recent progress, these techniques are not all effective, and unfortunately unfortunately frequently cause intolerance on the part of patients, as well as severe side effects such as bone marrow depression, vomiting, nausea, alopecia, asthenia, loss of appetite, anemia, thrombocytopenia, or non-preservation of healthy tissue.

For example, stage 4 neuroblastoma is treated intensively with chemotherapy, with significant short and long term side effects. Only 40% of patients recover, the others presenting successive relapses with a fatal outcome. Relapse cases are not predictable, and no therapy is currently available to treat them.

Thus, there is currently a need for methods making it possible to better predict the evolution of a tumor, in particular to determine its degree of aggressiveness and invasiveness, its probability of relapse after conventional treatment, and new, more effective anti-cancer active agents, which act selectively on cancer cells and their signaling pathways, which are less toxic, and which cause fewer, if any, or very few side effects.

Therapeutic targets specific to cancer cells are therefore actively sought, on the one hand, to distinguish tumors according to their stage, and on the other hand to identify new treatments targeting the different subtypes of neuroblastomas and which are better tolerated by patients.

In order to identify such targets, proteomics studies have shown that certain proteins are over-expressed or, on the contrary, under-expressed in neuroblastoma tumors, when these become metastatic. The level of expression of these proteins, or their associated genes, is therefore a reliable indicator of the severity and aggressiveness of the tumor.

In addition, the expression rate of several genes coding for these specific proteins makes it possible to define a "molecular signature" of the tumor, to anticipate its future, and therefore to adapt anti-cancer treatment.

Finally, proteins identified as biomarkers can be used as therapeutic targets. In particular, the proteins overexpressed in aggressive metastatic tumors can be advantageously regulated by means of specific inhibitors of their expression, which leads the tumor cells to adopt a less aggressive profile.

In the case of neuroblastomas, several specific targets have been identified so far and are under therapeutic investigation, but very few have led to encouraging preclinical results.

An overexpression of the anti-apoptosis factor Bcl-2 has been demonstrated in neuroblastic tumors; inhibition of the expression of this factor results in massive apoptosis of tumor cells treated with such an inhibitor, validating the interest of this therapeutic target (Lamers et al., 2012)

Overexpression of the Lin28B oncogene has been observed in aggressive neuroblastoma cells: this compound acts by negatively regulating the quantity of let-7 family microRNAs, which leads to a high level of N-MYC protein in neuroblastic cells, inducing aggressiveness of the tumor. (Molenaar et al., 2012)

Other biomarkers and therapeutic targets must now be identified, in order to develop new therapeutic options for patients with neuroblastoma, and to better distinguish the different types of tumors, other than by clinical exploration. The object of the present invention is to satisfy these needs.

STATEMENT OF THE INVENTION

The present invention relates to methods of diagnosing and treating neuroblastoma.

The present invention relates in particular to the identification of proteins, all members of the same cell signaling pathway, the expression level of which, considered for one or more of these proteins, varies according to the stage of the tumor, in particular when cancer cells progress from a "non-invasive" stage to an invasive, more aggressive stage.

More particularly, the present invention relates to a method for in vitro molecular characterization of a neuroblastoma tumor, comprising the following steps:

a) Obtaining a sample of said tumor;

b) Measurement in this sample of the level of expression of one or more of the 19 genes coding for the proteins chosen from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2,

c) Comparison of the level of expression of said gene (s) measured in step (b) with a reference value determined for the or each of said genes.

The present invention also relates to a modulator of the expression or of the activity of at least one protein chosen from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2, for use in the treatment of neuroblastoma.

More particularly, the present invention relates to:

Sema3C, or an activator of Sema3C expression or activity, for use in the treatment of metastatic stage 4 or 4S neuroblastoma; and

An inhibitor of the expression or activity of one or more plexins chosen from Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1 and Plexin D1, for its use in treatment of neuroblastoma.

DESCRIPTION OF THE FIGURES

Figure 1. For each biomarker gene coding for the protein mentioned at the top of the figure, a graph represents the expression levels of the gene measured in several tumor samples, from a cohort of patients (Kocak and Wolf). Similar results were obtained on a second cohort of patients (Shi and Fisher). The results are presented in the following order: stage 1 (st1), stage 2 (st2), stage 3 (st3), stage 4 (st4) and stage 4S (st4S) tumors. The numbers in parentheses indicate the number of samples for each stage. The expression rates are reported on the ordinate.

If a stage is distinguished from the others by a significantly different mean / median expression rate, the legend indicates: Different stage / other stages.

A correlation curve is presented at the bottom of the figure, representing a Kaplan-Meier curve making the link between the rate of expression of the biomarker gene and the rate of survival without relapse of the patients. The existence of a correlation between the rate of expression of the gene and a poor prognosis for survival is indicated below each graph.

A) Stage 4 specific biomarkers.

B) Specific biomarker of metastatic stages 4 and 4S.

C) Specific 4S stage biomarkers.

Figure 2. Genetic signature of the neuroblastic cell lines SHEP and IGRN91.

The expression levels of the genes coding for the proteins Plexin A1, Plexin A2, Neuropilin 1, Neuropilin 2, Sema3F, Sema3D, Sema3C, Sema3B and Sema3A were measured in immortalized cell lines derived from stage 4 neuroblastic tumors. of expression were normalized with respect to that of the HPRT gene.

Figure 3. Measurement of the expression variation factor of the genes coding for the proteins Sema3C, Sema3F and Sema3D, and comparison with a reference value

The reference value "0" represented in the figure corresponds to a zero variation between the level of expression measured in IGR-N91 cells before their transplant in a chicken embryo, and the level of expression measured in IGR-cells N91 after transplant and 48h incubation of the transplanted embryo.

The differences observed are represented in positive value (increase in the rate of expression) or negative (decrease in the rate of expression) compared to the reference value of the "naive" cells before their transplant in the embryo.

Figure 4. Effects of the introduction of interfering RNA directed against an inert scramble sequence: scr (control: CTL), against plexin A1 (PIxnAI) and against plexin A2 (PlxnA2)

A) The interfering RNAs are introduced into cells of neuroblastic lines SHEP and IGR-N91. Cell proliferation is measured using the Ce // Proliferation Reagent WST-1 kit (Merck).

B) The three interfering RNAs are introduced into SHEP neuroblastic cells. The activation of caspase-3 is measured by the DEVD-AFC technique which consists in measuring the fluorescence emitted by the cleavage of the DEVD-AFC compound by activated caspase-3.

C) Effects of the inhibition of the expression of plexin A1 (ΡΙχηΑΊ) and of plexin A2 (PlxnA2) by use of interfering RNAs (siPLxnAI and siPlxnA2) in a growth test in soft agar on SHEP neuroblastic cells , using as control an interfering RNA directed against an inert sequence (siCTL). The clones are visualized using the fluorescence emitted by GFP, expressed stably in SHEP cells.

D) Measurement of the size and of the number of clones formed in soft agar after inhibition of the expression of Plexin A1 and of Plexin A2

Figure 5. Inhibition of the expression of Plexins A1 and A2 with microRNAs of the miR-30 family in polycistronic form (“poly”, including mir-30a, mi-30b, mir-30c ') and with microRNA mir- 30C alone ("c")

HeLa cells were co-transfected with genetic constructs coding for the target genes (ΡΙχηΑΊ, PlxnA2 and Lin28b) integrated into a plasmid reporter for miRNA activity (pmiR-GIo luciferase reporter System, promega) and with the constructs coding for mir-30 in polycistronic form or for mir-30C alone. The expression of these fusion proteins is then evaluated as a function of the fluorescence activity measured on the cells.

Figure 6. Pro-metastatic role of Sema3C

A) Genetic construction making it possible to induce, in the presence of doxycycline, the expression of an sh-RNA directed against the messenger RNA of Sema3C. The corresponding results are presented in Table 3.

B) The IGR-N91 cells, after transient introduction of siRNAs directed against Sema3C, or of a control construct, are cultured in the form of drops, in a culture medium with or without Sema3C.

The cells are cultured in vitro in a control medium (on the left of the figure) or in a medium containing the protein Sema3C. The percentage of cell aggregation is indicated on the ordinate curve. The black dots represent the percentage of aggregation of cells transformed with a control interfering RNA (siRNA scr). The gray dots represent the percentage of aggregation of the cells transformed with an interfering RNA of sh-RNA type directed against the messenger RNA of sema3C (siRNA Sema3c).

DETAILED DESCRIPTION OF THE INVENTION

Preliminary remarks

The present invention is based on the identification of biomarker genes, coding for proteins involved in the signaling pathway of semaphorins, whose expression level is different from one tumor to another, and this difference in level of expression relative to a reference value being representative of the stage of the tumor.

The Semaphorin / Neuropilin / Plexin signaling pathway consists of a network of functionally associated genes, coding for secreted and / or transmembrane receptors (neuropilins and plexins) and ligands (semaphorins), whose interactions mediate migration controls and cell proliferation of neuroblastoma cells.

Semaphorins were first identified as factors guiding the progression of axons. This family of proteins includes more than 30 members, divided into 8 classes: classes 3 to 7 grouping the semaphorins expressed in vertebrates. All semaphorins are characterized by the presence of a ‘sema’ domain of about 500 amino acids, located near the N-terminal end, essential for the activity of these proteins. Semaphorins of class 3 (Sema 3A, 3B, 3C, 3D, 3E and 3F) are the only ones to be secreted and non-transmembrane in vertebrates.

The plexin family includes nine transmembrane receptors, characterized by the presence in their extracellular domain of a “sema” domain of around 500 amino acids, similar to that present in proteins of the semaphorin family. Plexins are the semaphorin receptors, in some cases in association with neuropilins, and transmit their signal via activation of intracellular pathways. The plexins all have, in their intracellular domain, a GAP domain (GTPase-activating protein) separated in two by an RBD domain (RHO-GTPasebinding domain). The dimerization of plexins would activate the GAP domain.

Four classes of plexins have been defined (A-D) according to their structure, and groups together the nine members of the family called A1, A2, A3, A4, B1, B2, B3, C1 and D1.

Each plexin has a specificity for the attachment of one or more semaphorins. For example, plexins A are the receptors for semaphorins 6: plexin A1 specifically fixes Sema6D, while plexins A2 and A4 bind the semaphorins Sema6A and Sema6B indifferently. All plexins A are capable of binding Sema3A. Plexin A1 binds Sema3E.

First identified for their role in the development of the nervous system, it was then shown that plexins are involved in embryonic development and in particular in processes such as angiogenesis, heart development, skeletal morphogenesis and morphogenesis kidney. (For review, see Peràlà et al., 2012).

The ‘NeuropilineT and‘ Neuropiline2 ’receptors (NPR1, NPR2) act as co-receptors for semaphorins, in association with plexins which transmit the signal to the cell by activating their GAP domain.

Studies on the functional involvement of this signaling pathway, in the context of the development of the nervous system, show that the interactions contracted between members of this pathway regulate cell behavior. Thus, the co-expression of ligands and receptors by the same cell modulates the response sensitivity of this cell to secreted semaphorins, present in the extracellular environment. For example, the couple Sema3A / Neuropiline1 expressed by a cell decreases the response of this cell to exogenous Sema3A (Moret et al, 2007). Similarly, the Sema3C / Neuropiline pair regulates the response to exogenous Sema3A, Sema3C and Sema3F. It decreases the sensitivity of the cell to the first two and on the contrary strengthens the response to the third (Sanyas et al, 2012). Generally speaking, there is a control of the level of expression of neuropilins on the cell surface, which determines the sensitivity of cells to ligands. In addition, the co-expression of plexins A and their transmembrane semaphorin ligands inhibits the cellular response to exogenous semaphorins. Finally, it has also been shown that, as a function of the members of the pathway expressed, and of its signaling effectors within the cell, the final functional effect can be a promoter effect or, on the contrary, an inhibitor of proliferation and cell migration. These data explain that in the context of cancers, the members of this signaling pathway are sometimes identified as oncogenes, or on the contrary are considered as tumor suppressors. Mutations in the genes of this signaling pathway, such as sema6C and plexin A1, for example, have recently been described in the genome of neuroblastoma tumors (Li et al., 2017).

Method for molecular characterization of a neuroblastoma tumor

The present invention relates to a method for the in vitro molecular characterization of a neuroblastoma tumor, comprising the following steps:

a) Obtaining a sample of said tumor;

b) Measurement in this sample of the level of expression of one or more of the genes coding for the proteins chosen from the group consisting of: a semaphorin, a plexin and a neuropilin,

c) Comparison of the level of expression of said gene (s) measured in step (b) with a reference value determined for the or each of said genes.

It is understood that the present invention relates to a neuroblastoma tumor, and that the semaphorins, plexins and neuropilins concerned are de facto those expressed by neuroblastoma cells.

Thus, the present invention relates to a method of molecular characterization in vitro of a neuroblastoma tumor, comprising the following steps:

a) Obtaining a sample of said tumor;

b) Measurement in this sample of the level of expression of one or more of the 19 genes coding for the proteins chosen from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2,

c) Comparison of the level of expression of said gene (s) measured in step (b) with a reference value determined for the or each of said genes.

Within the meaning of the invention, a molecular characterization process designates an in vitro process making it possible to obtain various information on the tumor: stage, risk of dissemination in the organism, aggressiveness, resistance to certain therapeutic treatments, etc. , this characterization being independent of the clinical examination.

The tumor samples are obtained according to the usual techniques, by qualified experimenters. In particular, samples can be taken from patients in hospitals or clinics, in oncology departments. It is however specified that this sampling step is excluded from the process according to the invention.

The measurement of the expression rate of a gene can be carried out according to all the conventional techniques well known to those skilled in the art. For example, commonly used techniques are qRT-PCR (quantitative Real Time - Polymerase Chain Reaction) and in situ hybridization on tissue sections and on cells.

The expression of the gene can also be determined via the assay of its product, the protein. The techniques for quantitative measurement of the proteins expressed are in particular the enzyme-linked immunosorbent assay (ELISA) technique for circulating proteins, immunohistochemistry on tissue sections or cells, and the Western blot for protein quantification on lysates and / or fluids.

The present invention also relates to a method for the in vitro molecular characterization of a neuroblastoma tumor, comprising the following steps:

a) Obtaining a sample of said tumor;

b) Measurement in this sample of the production rate of one or more of the 19 proteins chosen from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2 , Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2,

c) Comparison of the production rate of said protein (s) measured in step (b) with a reference value determined for the or each of said proteins.

This characterization process may be carried out in place of that comprising the measurement of the gene expression rate, or else be carried out in addition to the process comprising the measurement of the gene expression rate.

Furthermore, within the meaning of the invention, the measurement of the rate of production of one or more proteins includes the measurement of the rate of presence on the surface of membrane proteins or proteins associated with the membrane, and / or the rate of secretion of proteins by the cell.

In particular, the rate of presence on the surface of membrane proteins or associated with the membrane, and the quantity of proteins secreted by the cell, can be compared, which will give additional information on the molecular characterization of the tumor.

The expression “reference value” designates the quantified value of a rate of expression of a gene in one or more tumors of which the stage (s) is (are) known. For a gene (i), this value will be designated below Vi.

According to a preferred aspect of the invention, the reference value Vi is an average or a median of several values of the rate of expression of the same gene, measured in different tumors of known stage.

It is thus possible to define a reference value for each gene, specific for stage 1 tumors, specific for stage 2 tumors, specific for stage 3 tumors, specific for stage 4 tumors and specific for stage 4S tumors. Other reference values can be defined, as will be presented later.

The comparison of the value obtained in step (b) for a gene (i), this value being designated hereinafter Ti, with the corresponding reference value Vi, makes it possible to determine whether or not there is a difference between these two values.

In the present application, the terms “difference” and “variation” are used interchangeably, and both designate either a significant increase, or a significant decrease, in the value of the level of expression of a Ti gene relative to its reference value Vi.

Within the meaning of the invention, a difference (increase or decrease) is considered to be significant by carrying out statistical tests well known to those skilled in the art.

According to one embodiment of the invention, the reference values and measured expression rates can be normalized, to take account of the quantity of cellular biomass. This normalization of the relative values obtained for each gene is obtained by dividing these values by the value of the rate of expression of a messenger RNA representative of the quantity of cells. The messenger RNA representative of the quantity of cells can in particular be that of the protein HPRT (hypoxanthine-guanine phosphoribosyltransferase). This same technique for normalizing relative values can be implemented when the rates measured are rates of protein production.

According to a particular implementation of the invention, the method further comprises a step d) of determining the stage of the tumor from one of the stages 1, 2, 3, 4 or 4S, as a function of the difference observed between the level of Ti expression of at least one gene as mentioned above, and its reference value Vi.

The expression “stage determination” designates a particular aspect of the molecular characterization of the tumor, consisting in attributing to the tumor a classification among the following categories: stages 1,2, 3, 4 or 4S, these clinical stages having been defined by hospital practitioners. However, the method according to the invention here implements a technology independent of conventional clinical investigations.

Advantageously, the method according to the invention can be carried out by measuring, in the tumor sample, the level of expression of a single gene chosen from those coding for Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2.

Indeed, each biomarker can be used independently of the other biomarkers to molecularly characterize the tumor from which the studied sample comes.

However, it may be relevant to measure the level of expression of several genes coding for proteins chosen from semaphorins, plexins and neuropilins, in order to obtain a "map" also called "molecular signature" of a tumor.

Such a molecular characterization of a tumor allows the experimenters to determine, as a function of the differences observed with the reference values Vi for each gene (i), as "precisely" as possible the "status" of the tumor, this including the stage, the risk of metastasis, aggressiveness, invasiveness and the chances of response to a treatment for said tumor. For tumors with the same characteristic, variations in this signature are representative of differences between patients.

Advantageously, the molecular characterization of a tumor according to the method of the invention can allow healthcare personnel to choose the therapeutic treatment most suited to the tumor thus characterized, and thus to practice personalized medicine.

When the expression level of several genes is measured, it is understood that the expression level may be measured for two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen , fifteen, sixteen, seventeen, eighteen or nineteen genes chosen from the genes coding for the proteins Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2 , Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2.

It is understood that all possible combinations of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen or nineteen selected genes among the genes coding for the proteins Sema3C, Sema3A, Sema3B,

Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2 are included according to the invention.

Among all these combinations of 19 genes, it will in particular be possible to measure the level of expression of the genes coding for the following proteins:

at least one semaphorin, at least one plexin and at least one neuropilin;

at least one semaphorin and at least one plexin;

at least one semaphorin and at least one neuropilin;

at least one semaphorin;

at least Sema3C;

at least two semaphorins and at least two plexins;

at least Sema3D, Sema4C, Plexine A3 and Plexine B1;

at least three semaphorins, at least four plexins and at least one neuropilin;

at least Sema4C, Sema4D, Sema6D, Plexine A3, Plexine A4, Plexine B2, Plexine C1, Plexine D1 and Neuropiline 1.

According to a first aspect of the invention, in step (b), the expression level is measured for one or more of the 9 genes coding for the following proteins: Sema4C, Sema4D, Sema6D, Plexin A3, Plexin A4 , Plexin B2, Plexin C1, Plexin D1 and Neuropilin 1.

This particular combination of nine biomarkers is particularly indicated for distinguishing stage 4 tumors, as presented in FIG. 1 A. Indeed, all of these genes exhibiting a significantly different level of expression than those found in samples of tumor of 1,2, 3 or 4S stage.

In this implementation, the reference value Vi for each gene is obtained from measurements of expression rate of the gene (i) on several tumors of which the stage 1,2, 3 or 4S is known.

The reference value Vi may in particular be the result of the arrhythmic mean, or of the median, calculated from the expression rates obtained on tumors of which stage 1,2, 3 or 4S is known.

According to a specific implementation, the present invention relates to a method of in vitro characterization of a neuroblastoma tumor, in which:

- In step (b), the expression level is measured for one or more of the 9 genes coding for the following proteins: Sema4C, Sema4D, Sema6D, Plexine A3, Plexine A4, Plexine B2, Plexine C1, Plexine D1 and Neuropiline 1, and

- In step (c), for each gene, the reference value is a median of the expression rate of said gene measured in tumors of clinical stage 1,2, 3 and 4S.

According to another specific implementation, the present invention relates to an in vitro method for determining the stage of a neuroblastoma tumor, comprising the following steps:

a) Obtaining a sample of said tumor;

b) Measurement in this sample of the level of expression of one or more of the 9 genes coding for the proteins chosen from the group consisting of: Sema4C, Sema4D, Sema6D, Plexin A3, Plexin A4, Plexin B2, Plexin C1, Plexin D1 and Neuropilin 1, and

c) Comparison of the level of expression of said gene or genes measured in step (b) with a reference value determined for the or each of said genes, this reference value being a median of the level of expression of said gene measured on tumors of clinical stage 1,2, 3 and 4S.

According to this implementation, in cases where a significant difference is observed between the level of Ti expression of a gene in the tumor whose stage must be determined, and its reference value Vi, this will mean that the tumor is stage 4.

According to a second aspect of the invention, in step (b), the expression rate is measured for one or more of the 3 genes coding for the following proteins: Sema4C, Sema3D and Plexin B1.

This particular combination of three biomarkers is particularly indicated for distinguishing stage 4S tumors, as shown in Figure 1C. Indeed, all these genes having a significantly different level of expression than those found in samples of stage 1,2, 3 or 4 tumors.

In this implementation, the reference value Vi for each gene is obtained from measurements of expression rate of the gene (i) on several tumors of which stage 1, 2, 3 or 4 is known.

The reference value Vi may in particular be the result of the arrhythmic mean, or of the median, calculated from the expression rates obtained on tumors of which stage 1,2, 3 or 4 is known.

According to a specific implementation, the present invention relates to a method of in vitro characterization of a neuroblastoma tumor, in which:

- In step (b), the expression rate is measured for one or more of the 3 genes coding for the following proteins: Sema4C, Sema3D and Plexin B1, and

- In step (c), for each gene, the reference value is a median of the expression rate of said gene measured in tumors of clinical stage 1, 2, 3 and 4.

According to another specific implementation, the present invention relates to an in vitro method for determining the stage of a neuroblastoma tumor, comprising the following steps:

a) Obtaining a sample of said tumor;

b) Measurement in this sample of the level of expression of one or more of the 3 genes coding for the proteins chosen from the group consisting of: Sema4C, Sema3D and Plexin B1, and

c) Comparison of the level of expression of said gene or genes measured in step (b) with a reference value determined for the or each of said genes, this reference value being a median of the level of expression of said gene measured on tumors of clinical stage 1,2, 3 and 4.

According to this implementation, in cases where a significant difference is observed between the level of Ti expression of a gene in the tumor whose stage must be determined, and its reference value Vi, this will mean that the tumor is stage 4S.

According to a third aspect of the invention, in step (b), the measurement of the expression level is carried out for at least the gene coding for the protein Sema3C.

The use of this biomarker is particularly indicated to distinguish tumors of stage 4 or 4S, that is to say tumors of the metastatic type, as is presented in FIG. 1 B. Indeed, this gene exhibits a rate significantly different in expression (its expression is significantly reduced) than those found in samples of stage 1,2 and 3 tumors.

In this implementation, the reference value Vi for this gene is obtained from measurements of the level of expression of the gene coding for Sema3C on several tumors of which stage 1,2 or 3 is known.

The reference value Vi may in particular be the result of the arrhythmic mean, or the median, calculated from the expression rates obtained on tumors of which stage 1,2,3 is known.

According to a specific implementation, the present invention relates to a method of in vitro characterization of a neuroblastoma tumor, in which:

- In step (b), the expression rate is measured for at least the gene coding for the protein Sema3C; and

- In step (c), the reference value is a median of the expression rate of said gene measured in tumors of clinical stage 1, 2 and 3.

According to another specific implementation, the present invention relates to an in vitro method for determining the stage of a neuroblastoma tumor, comprising the following steps:

a) Obtaining a sample of said tumor;

b) Measurement in this sample of the expression level of at least the gene coding for the protein Sema3C, and

c) Comparison of the level of expression of the gene measured in step (b) with a reference value determined for the gene coding for Sema3C, this reference value being a median of the level of expression of said gene measured on tumors of clinical stage 1, 2, and 3.

According to this implementation, in cases where a significant difference is observed between the level of expression T of the sema3C gene in the tumor whose stage must be determined, and its reference value Vi, this will mean that the tumor is of stage 4 or 4S and is therefore at a metastatic stage.

Method for monitoring a neuroblastoma tumor

The method according to the present invention advantageously makes it possible to follow the progress of a tumor in a given patient.

For this, the in vitro molecular characterization process of a tumor will be carried out on samples from the same tumor, but obtained at different times, in particular before and after the start of a therapeutic treatment, or before and after an intervention. surgery, or even after remission and then during a tumor recurrence.

More generally, step (a) of obtaining the tumor sample may be carried out at a time and the reference value will be that obtained by measuring the level of expression of a gene on a tumor sample obtained. at a time T _o , T _o being prior to

According to this implementation, the in vitro molecular characterization process of the tumor is characterized in that:

step (a) of obtaining a sample of a patient's tumor is carried out at a time Ti; and

- In step (c), the reference value determined for a gene is the level of expression of said gene measured in a tumor sample from the same patient, obtained at a time T _o ,

T _o being before

According to a particular implementation of the method, the time Ti is located chronologically after the start of the administration of a therapeutic treatment to the patient; and the time T _o is located chronologically before the start of said treatment.

Thus, the comparison of the molecular profile of the tumor before and after the start of treatment makes it possible to assess the therapeutic effects of the treatment, and possibly to adapt it to improve its effects.

According to a specific implementation, the present invention relates to an in vitro method for monitoring the progress of a neuroblastoma tumor in a patient, comprising the following steps:

a) Obtaining a sample of said tumor at a time Ty

b) Measurement in this sample of the level of expression of one or more of the 19 genes coding for the proteins chosen from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2,

c) Comparison of the expression level of said gene or genes measured in step (b) with a reference value determined for the or each of said genes, said reference value determined for each gene having been obtained by measuring the level d expression of said gene in a tumor sample from the same patient obtained at a time T _o , T _o being before Tv

DNA chips and diagnostic kits

The invention also relates to a DNA chip carrying at least 19 oligonucleotide probes, each of the probes being specific for a gene from the genes coding for the following proteins: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2.

The present invention also relates to a diagnostic kit making it possible to implement the method as described above, comprising the following components:

A DNA chip as described above, and

Reagents for quantifying, in a neuroblastoma tumor sample, the level of expression of at least one gene chosen from the genes coding for the following proteins: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D , Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2.

The invention also relates to the use of this DNA chip and / or of this diagnostic kit for in vitro characterization of a neuroblastoma tumor, from a molecular point of view.

The invention also relates to the use of this DNA chip and / or of this diagnostic kit for monitoring in vitro the progress of a neuroblastoma tumor in a patient.

Therapeutic compounds for use in the treatment of neuroblastoma

The concept of "treatment of neuroblastoma" covers several aspects such as the reduction of the size of the tumor, the disappearance of the primary tumor and / or secondary tumors (metastases) and the prevention of recurrences.

The term “treating cancer” is understood to mean slowing down or completely inhibiting the development of a tumor in the patient, and / or making completely disappear from the patient's body all the cancer cells and in particular the tumor of 'origin.

The present invention describes therapeutic targets for the treatment of neuroblastoma: these are the proteins Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4 , Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2.

The term “therapeutic compound” means a compound which is effective in slowing down or inhibiting the development of a neuroblastoma tumor in the patient, and / or a compound which makes it possible to remove a neuroblastoma tumor. By “tumor” is meant a primary tumor or a secondary tumor (metastasis) resulting from the spread of the primary tumor.

More specifically, within the meaning of the invention, such a therapeutic compound consists of a modulator of the expression or of the activity of at least one protein chosen from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2.

The invention therefore relates to a modulator of the expression or activity of at least one protein chosen from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2, for use in the treatment of neuroblastoma.

The expression “expression or activity modulator” designates a compound capable of specifically influencing the expression or the activity of a protein chosen from the list above.

Depending on the targeted protein, it may be an activator or an inhibitor of said expression or activity.

Those skilled in the art will be able to choose from among the various compounds capable of exhibiting the required activity: for example, the inhibition of the expression of a protein may be carried out using interfering RNA or microRNAs; inhibition of its activity can be achieved by the use of antagonists, in particular antibodies; and stimulation of its expression can be obtained using stabilizing compounds for messenger RNA molecules.

It should be noted that the sequences of these proteins, in particular human proteins, as well as the sequences of the genes encoding these proteins, are accessible in public databases and can be used to generate compounds that inhibit or activate expression or of the activity of said proteins.

The present invention also relates to a method for treating neuroblastoma, comprising the administration to a patient suffering from this cancer of a modulator of the expression or of the activity of at least one protein chosen from the group consisting of: Sema3C , Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2.

The invention also relates to Sema3C, or an activator of the expression or activity of Sema3C, for its use in the treatment of metastatic stage 4 or 4S neuroblastoma.

Indeed, the inventors have identified the pro-metastatic role of a decrease in the expression of Sema3C: as shown in FIG. 6B, and in Table 3, in the event of a decrease in the expression of Sema3C, the cells which previously had a cohesive behavior disintegrate and migrate to other tissues. The results presented in FIG. 6B demonstrate that the cohesion of these cells can be restored by adding an exogenous Sema3C protein.

Thus, the Sema3C protein or any compound capable of activating its expression or activity, or even any compound capable of mimicking its activity, are likely to be used in the treatment of metastatic stage neuroblastoma.

It is understood that the term "Sema3C" designates both the protein as such, but also its derivative forms as well as peptides or agonists mimicking the activity of this protein, and also includes the nucleic acids coding for a protein

Sema3C, human or not, or their counterparts coding for a protein of another species, having the same biological function.

The invention also relates to a method for treating metastatic stage neuroblastoma, and / or for preventing metastasis in a patient with non-metastatic stage neuroblastoma tumor, comprising administering to said patient a Sema3C protein, or an activator of the expression or activity of Sema3C.

The present invention also relates to an inhibitor of the expression or of the activity of at least one plexin chosen from Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1 and Plexin D1, for its use in the treatment of neuroblastoma.

According to a particular implementation, the present invention relates to an inhibitor of the expression or of the activity of at least one plexin chosen from Plexin A1 and Plexin A2.

More particularly, the present invention relates to an inhibitor of the expression or activity of Plexin A1.

More particularly, the present invention relates to an inhibitor of the expression or activity of Plexin A2.

The present invention also relates to a method for treating neuroblastoma, comprising the administration to a patient suffering from this cancer of an inhibitor of the expression or of the activity of at least one plexin chosen from Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1 and Plexin D1.

According to a first embodiment of the invention, the inhibitor of at least one plexin is an inhibitor of its activity.

An inhibitor of the activity of a plexin is a compound which acts on one of the activities below, by opposing it:

(i) the capacity of plexin to form a homodimer, (ii) its capacity to bind its natural ligand (s), (iii) its capacity to activate one of the intracellular signaling pathways following fixation of its natural ligand (s).

An inhibitor acting on the ability of plexin to form a homodimer will in particular be a protein molecule, in particular a peptide or an antibody, capable of modifying the spatial conformation of plexin in order to prevent its dimerization with another plexin.

An inhibitor acting on the ability of plexin to bind its natural ligand (s), such as a semaphorin, will in particular be a protein molecule, in particular a peptide or an antibody, capable of modifying the spatial conformation of the plexin to prevent the binding of semaphorin, and / or capable of blocking or sterically obstructing the site of attachment of semaphorin.

An inhibitor acting on one of the intracellular signaling pathways activated by a plexin will in particular be a protein molecule, in particular a peptide or an antibody, capable of modifying the spatial conformation of plexin in order to prevent the activation of the catalytic domains present in the intracellular domain of plexin, or blocking the binding site to intracellular proteins involved in this signaling pathway.

It is understood that the same inhibitor of the activity of at least one plexin may act on several capacities of said plexin, such as for example on its capacity to form a homodimer and to bind its natural ligand.

According to a particular implementation of the invention, the inhibitor of the activity of at least one plexin is chosen from an antibody or a peptide.

According to a second embodiment of the invention, the inhibitor of at least one plexin is an inhibitor of its expression.

Those skilled in the art know many ways of reducing the expression of a protein, which include, but are not limited to, all technologies of genetic modification, and all factors inhibiting the transcription of the gene coding for a plexin, or even translation. messenger RNA to protein; these means include the use of interfering RNAs and microRNAs.

Such an inhibitor of plexin expression will in particular be an inhibitor which effectively reduces, that is to say at least 40%, or better at least 60%, or better still at least 80% , the expression of the plexin against which it is directed.

More particularly, the inhibitor of the expression or of the activity of at least one plexin can be an expression inhibitor chosen from an interfering RNA and a micro-RNA.

According to a particular implementation of the invention, the inhibitor is an interfering RNA inhibiting the expression of at least one plexin.

Interfering RNAs are RNAs which hybridize to messenger RNA (mRNA), a product of expression of the gene comprising the sequence coding for plexin. The interfering RNAs inhibit translation either by simple steric hindrance or by promoting the cleavage of the mRNA.

Interfering RNA technologies and their use in vitro and in vivo are well known to those skilled in the art, and are described in numerous scientific articles and patent applications. Preferably, the interfering RNAs are prepared and selected to obtain at least 40% inhibition of the expression of the target gene in a cell, or even at least 50%, 60%, 70%, 80%, 90%, 95 % or even more than 99% inhibition.

Among the interfering RNAs, the “siRNA” for Small Interfering RNA or small interfering RNA, are short sequences of approximately 15 to 30 base pairs (bp), preferably from 19 to 25 bp. The design and preparation of siRNAs and their use for transfection of cells in vivo and in vitro are well known and described in numerous publications.

SiRNAs can be designed and prepared using suitable software available online, well known to those skilled in the art.

The siRNAs can also be ordered from specialized commercial companies, such as Santa Cruz and Sigma-Aldrich, which supply pre-designed siRNAs on request.

Among the interfering RNAs, mention will also be made of “shRNA” or small hairpin RNAs, which are RNAs adopting a rod and loop structure.

The present invention also relates to a vector for the expression of an interfering RNA defined above and below, which vector comprising a nucleic acid comprising a sequence coding for said interfering RNA placed under the control of one or more elements of regulation allowing the expression of said interfering RNA in a host cell.

The invention also relates to a vector for the delivery of an RNA interfering in a host cell, characterized in that it comprises an RNA interfering according to the invention, defined above and below and means allowing the delivery of said RNA interfering in a host cell. Such means are known to a person skilled in the art, such as, for example, lipofectamine lipids (http://www.invitrogen.com/content.cfm?pageid=4005) or Mi ru s (http: //www.mirusbio. com / products / RNAi / index.asp).

Interfering RNA capable of inhibiting the expression of certain plexins have been described in the prior art, and are incorporated into the present application by reference.

We can cite in particular:

interfering RNAs inhibiting the expression of plexin A1 were used to elucidate the signaling pathways activated in response to the binding of semaphorin Sema6D (Catalano et al., 2009);

interfering shRNA RNAs inhibiting the expression of plexins A1, A2, A3 and A4 have been used in HUVEC endothelial cells (Kigel et al., 2011);

interfering RNAs of the shRNA type inhibiting the expression of plexin A1 have been introduced into gastric cancer cells MGC803, causing a decrease in the levels of VEGFR2 (Lu et al., 2016);

interfering shRNA RNAs that inhibit plexin A1 expression have been used in lung carcinoma cells (Yamada et al., 2016).

According to another particular implementation of the invention, the inhibitor is a micro-RNA inhibiting the expression of at least one plexin.

MicroRNAs, abbreviated as miRNAs, are short, single-stranded ribonucleic acids (RNAs) (21-24 nucleotides), expressed only in eukaryotic cells.

MiRNAs are post-transcriptional regulators capable of inhibiting the expression of a gene: their pairing with a sequence complementary to the messenger RNA of the target gene leads to translational repression or to the degradation of this messenger RNA.

In humans, microRNAs are abundant in a large number of cell types, and are said to target around 60% of genes. Thus, miRNA-based therapies are currently being studied, these therapies aiming to specifically inhibit the expression of certain genes, in particular involved in tumor processes.

Thus, the inhibition of the expression of at least one plexin can be achieved by the use of at least one micro-RNA specifically targeting this plexin.

According to a preferred aspect of the invention, the microRNA used for the treatment of neuroblastoma is a microRNA of the miR-30 family.

The expression of the miR-30 family is very greatly reduced in aggressive neuroblastomas, suggesting that these could regulate the tumor growth of neuroblastomas.

The results of the experiments carried out by the inventors, presented in FIG. 5, demonstrate that this microRNA has a clear effect on the expression of plexins A1 and A2, the inhibition of expression being significant and being accompanied by a functional effect since the Neuroblastoma cells exhibit decreased proliferation when the expression of plexins is thereby inhibited.

The present invention also relates to a vector for the expression of a micro-RNA as defined above, which vector comprising a nucleic acid comprising a sequence coding for said micro-RNA placed under the control of one or more elements of regulation allowing the expression of said micro-RNA in a host cell.

The invention also relates to a vector for the delivery of a micro-RNA into a host cell, characterized in that it comprises a micro-RNA as defined above, and means allowing the delivery of said micro-RNA in a host cell.

The present invention also relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable medium, at least one therapeutic compound chosen from the group consisting of:

a modulator of the expression or the activity of a protein chosen from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3 , Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2,

Sema3C or an activator of the expression or activity of Sema3C, and an inhibitor of the expression or activity of at least one plexin of at least one plexin chosen from Plexin A1, Plexin A2, Plexin A3 , Plexin A4, Plexin B1, Plexin C1 and Plexin D1.

An acceptable pharmaceutical medium denotes a vehicle making it possible to store and administer the inhibitor according to the invention, and optionally excipients, the administration of which to an individual or an animal is not accompanied by significant deleterious effects, and which are well known to those skilled in the art.

A pharmaceutical composition according to the invention can comprise any pharmaceutical excipient necessary, such as buffering agents or agents for adjusting the pH or isotonicity, or also wetting agents. A pharmaceutical composition according to the invention can also comprise one or more antioxidant agents, and / or one or more preservative agents.

A pharmaceutical composition as described above can be administered by any suitable route, such as the oral, buccal, sublingual, ophthalmic, rectal, topical route, or by the parenteral route, in particular by an intraperitoneal, intradermal, subcutaneous route. , intravenous or intramuscular.

A pharmaceutical composition according to the invention can be formulated for oral administration, in the form of a tablet, a capsule or a gelatin capsule, with sustained or controlled release, of a pill, of a powder , solution, suspension, syrup or emulsion.

According to another embodiment, a pharmaceutical composition according to the invention can be prepared for administration by the parenteral route, in an injectable form. A polypeptide or a nucleic acid as described in the invention can be formulated with different vehicles, such as a liposome or a transfection polymer.

A pharmaceutical composition of the invention may preferably comprise a nucleic acid, in particular an interfering RNA or a microRNA as defined in the present description, in suspension in a pharmaceutically acceptable vehicle, for example an aqueous vehicle. Different aqueous vehicles can be used, for example water, saline buffer solution, 0.4% or 0.3% glycine solution, or hyaluronic acid solution.

A pharmaceutical composition according to the invention can be sterilized by any known conventional method, such as filtration. The resulting aqueous solution can be packaged for use as it is, or can be freeze-dried. A lyophilized preparation can be combined with a sterile solution before use.

According to a preferred implementation of the invention, the pharmaceutical composition comprises an effective amount of a therapeutic compound as defined above.

An effective amount of a therapeutic compound is an amount which, alone or in combination with subsequent doses, induces the desired response, namely an antiproliferative effect on cancer cells. The effective amount of such an inhibitor may depend on one parameter or a plurality of parameters, such as the route of administration, single or multiple dose administration, patient characteristics, which includes age. , physical condition, height, weight and the presence of conditions in addition to that treated. These parameters and their influences are well known to those skilled in the art and can be determined by any known method.

Preferably, said pharmaceutical composition comprises as therapeutic compound an interfering RNA or a microRNA inhibiting the expression of at least one plexin chosen from Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1 and Plexin D1, and in particular a microRNA of the miR-30 family.

The present invention also relates to a combination product comprising:

at least one therapeutic compound chosen from the group consisting of:

o a modulator of the expression or activity of a protein chosen from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2, o Sema3C or an activator of the expression or activity of Sema3C, and o an inhibitor of the expression or activity at least one plexin at least one plexin selected from Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1 and Plexin D1, and at least one chemotherapy agent or an anti-angiogenic agent or a immunotherapy agent.

A chemotherapy agent suitable for the invention can be chosen from cytostatics, antimetabolites, DNA intercalating substances, topoisomerase I and II inhibitor compounds, tubulin inhibitors, alkylating agents, neocarcinostatin, calicheamicin , dynemicin or esperamicin A, ribosome inhibitor compounds, tyrosine phosphokinase inhibitors, cell differentiation inducing compounds, or histone deacetylase inhibitors.

In particular, a chemotherapy agent suitable for the invention can be chosen, without limitation:

• among cytostatics and antimetabolites, such as 5-fluorouracil, 5-fluoro-cytidine, 5-fluoro-uridine, cytosine arabinoside or methotrexate, • among DNA intercalating substances, such as doxorubicin , daunomycin, idarubicin, epirubicin or mitoxantrone, • among topoisomerase I and II inhibitor compounds, such as camptothecin, etoposide or m-AMSA, • among tubulin inhibitors, such as vincristine, Vinblastine, vindésine, taxol, nocodazole or colchicine, • among alkylating agents, such as cyclophosphamide, mitomycin C, rachelmycin, cisplatin, phosphoramide mustard gas, melphalan, bleomycin, N-bis (2-chloroethyl) -4-hydroxyaniline, • among neocarcinostatin, calicheamicin, dynemicin or esperamicin A, • among ribosome inhibitor compounds, such as verrucarin A, • among tyrosin inhibitors e phosphokinases, such as quercetin, genistein, erbstatin, tyrphostin or rohitukin and its derivatives, • among compounds which induce cell differentiation, such as retinoic acid, butyric aid, phorbol esters or aclacinomycin, or • among histone deacetylase inhibitors, such as CI-994 or MS275. An angiogenesis-inhibiting agent suitable for the invention can be chosen from the following non-limiting lists:

monoclonal antibodies targeting VEGF such as Bevacizumab, protein compounds mimicking domains of VEGF receptors, such as Aflibercept, tyrosine phosphokinase inhibitors such as Sorafenib, Sunitinib, Pazopanib, Axitinib, Vantetanib or Cabozantinib, analogs of rapamycin such as Temsirolimus or Everolimus.

An immunotherapy agent suitable for the invention can be chosen from cytokines, such as interleukin-2, cells of the immune system, in particular immune cells from the patient taken, activated and reinjected into the latter, or antibodies directed against cancer cells.

An inhibitor of the expression or activity of at least one plexin can also be used in combination with radiotherapy treatment.

A radiotherapy treatment which may be suitable for the invention may be external radiotherapy using an external source of X-rays, brachytherapy consisting in bringing the radioactive source into contact with the tumor, or vector metabolic radiotherapy. These methods are known to those skilled in the art.

The present invention also relates to a combination product as defined below, for its simultaneous, separate or sequential use in the treatment of neuroblastoma.

EXAMPLES

Table 1. List of genetic tools and their sources

Product Name Provider Reference siRNA Universal Negative Control # 1 Sigma-AldrichAldrich SIC001 human Sema3C siRNA (NM_006379) Sigma-AldrichAldrich SASI_Hs01_00161889 Human Sema3C # 2 siRNA (NM_006379) Sigma-AldrichAldrich SASI_Hs01_00161890 SMARTvector Inducible Non-targeting mCMVturboRFP Control Dharmacon VSC6571 SMARTvector Inducible Human SEMA3C mCMVTurboRFP shRNA Dharmacon V3SH7676-08EG10512 (shRNA sequence V3SH7670-225814784) human PlexinAI siRNA Sigma-AldrichAldrich SASI_Hs01_00010384 human PlexinA2 siRNA Sigma-AldrichAldrich SASI_Hs01_00153626 miR-30 polycistron N / A hsa-miR-30c-2 (acc number: MI0000254, HUGO :) hsa-miR-30d (MI0000255, MIR30D) hsa-miR-30a (MI0000088, MIR30A) hsa-miR-30e (MI0000749, MIR30E) hsa-miR- 30b (MI0000441, MIR30B)

The statistical analysis of the data was carried out with Prism 6.0e software (GraphPad).

EXAMPLE 1 Expression Profiles of the Semaphorin Signaling Pathway Genes in Samples of Neuroblastoma Tumors of Different Stages

The patient cohorts studied are presented in the following bibliographical references:

Cohort GSE45480 of Wolf & Kocak: Kocak et al., 2013; this cohort references 649 tumor samples;

Shi & Fisher cohort GSE49710: Wang et al., 2014; this cohort includes 498 tumor samples.

The Agilent-020382 44k microchip (GPL16876) was used. Bioinformatics analyzes were carried out with the Genomics Analysis and Visualization Platform (http://r2.amc.nl).

a) Stage 4 biomarkers

FIG. 1A presents the expression rate measurements carried out on samples of neuroblastic tumors, drawn from the patient cohort GSE45480 from Wolf & Kocak.

One or two probes were used for each gene.

The results obtained on the Shi & Fisher cohort GSE49710 (not shown) are identical to those presented here, and this for each of the genes studied.

Nine genes show significant differences in expression in stage 4 tumor samples compared to the expression measured in stage 1, 2, 3 and 4S tumors.

Seven of these genes are also correlated with a poor prognosis. Only the expression levels of plexins D1 and B2 are not linked to a poor survival prognosis.

b) Metastatic stages 4 and 4S biomarkers

FIG. 1B presents the measurement of the level of expression of the gene coding for Sema3C carried out on samples of neuroblastic tumors, originating from the cohort of Kocak patients (GSE45480).

The results obtained for the Kocak and Shi & Fisher cohorts are identical.

The sema3C gene shows a significant variation in expression in stage 4 and 4S tumor samples, compared to stage 1, 2 and 3 tumors. This is a decrease in the expression of Sema3C.

In addition, a correlation with a poor prognosis for survival is established.

c) Stage 4S specific biomarkers

Figure 1C presents the expression rate measurements carried out on samples of neuroblastic tumors, from the Kocak patient cohort (GSE45480)

The results obtained on the Shi & Fisher cohort are identical (not shown).

The genes encoding Sema3D, Plexin B1 and Sema4C show a significant difference in expression in samples of stage 4S tumors, compared to the expression rates observed in tumors of stages 1,2, 3 and 4.

The differences observed are as follows: for Sema3D, a decrease in gene expression in 4S tumors. and for Plexin B1 and Sema4C, a significant increase in expression in 4S tumors.

Example 2. Expression Profiles of Nine Genes of the Semaphorin Signaling Pathway in Immortalized Cell Lines Derived from Stage 4 Neuroblastoma Tumors

a) The level of expression of seven genes was measured in a panel of 26 primary lines and 4 immortalized lines derived from stage 4 neuroblastoma tumors. The expression quantified by quantitative RT-PCR (RT-qPCR) reveals differences expression levels for each gene, from one line to another. (Results not shown)

b) The expression level of nine genes was measured in immortalized cells from neurobastoma, the IGR-N91 and SHEP lines. The technique used is RT-qPCR, the amplified nucleic acid level being normalized compared to that of the gene coding for the household protein HPRT. Figure 2 shows these different levels of expression, which vary from one line to another.

These results highlight the expression of these nine genes by neuroblastoma cells.

It is understood that the expression rate of these genes presented here does not make it possible to deduce, with these data alone, that these genes can serve as biomarkers.

Indeed, it is the differences in expression observed between one stage and another that are significant with regard to a diagnostic or monitoring process.

EXAMPLE 3 Expression Profiles of Genes of the Semaphorin Signaling Pathway in Samples of Stage 4 Neuroblastoma Tumors, After Transplantation in HI-114 Chicken Embryos and Incubation for 48 Hours

Neuroblastoma tumors have been produced in vivo by micrografting human tumor cells into the tissues of an avian embryo, a tumorignesis model described in patent application WO 2016/005398.

Tumors form in sympathetic derivatives of the host, mimicking the site of primary tumors in patients with neuroblastoma.

The tumors are then removed and their transcriptome is compared by RNAseq (random sequencing of the entire transcriptome) with that of these same cells before their transplant ("naive tumors").

This analysis revealed an expression modification signature for 13 genes in the Semaphorin / Neuropilin / Plexin network, which correlates expression analysis data in patient cohorts.

a) Cells of the neuroblastic cell line IGR-N91 were grafted into a chicken embryo according to the protocol set out in patent application WO 2016/005398.

After 48 hours of incubation, the tumors that developed within the embryo were removed and analyzed. The expression levels of the genes coding for Sema3C, Sema3F and Sema3D were determined by quantitative RT-PCR, both on IGR-N91 cells before the transplant (“naive cells”), and on tumors removed from the chicken embryo 48 after this transplant.

FIG. 3 shows the variations in expression observed between the cells before their implantation in the embryo, and the cells which have formed a tumor in vivo in an animal model. The genes encoding Sema3C and Sema3F show a decrease in expression after implantation in vivo; on the contrary, the Sema3D gene has a strong increase in its expression rate. These data confirm the results obtained by RNASeq.

b) Cells from neuroblastoma tumors were grafted into a chicken embryo according to the protocol set out in patent application WO 2016/005398.

After 48 hours of incubation, the tumors that developed within the embryo were removed and analyzed. The expression levels of 13 genes were determined by extracting the results of random sequencing of the entire transcriptome, both on tumor cells before the transplant (“naive cells”), and on tumors removed from the chicken embryo. 48 after this transplant.

Table 2 below presents the differences in expression observed between the cells before their implantation in the embryo, and the cells which formed a tumor in vivo in an animal model.

Table 2. Variation in the expression rate of 13 genes of the semaphorin signaling pathway after tumor development in vivo in an animal model

Accession number (NCBI platform) Chromosome locus Gene encoding the protein: Average expression rate in sampled tumors Average expression rate in "naive cells" Variation between the two cell types p-value (Eqvar) NM_006080 CHR 7: 83587658-83824217 Sema3A 8.53902 6.96972 1.22516 0.439958 NM_006379 CHR 7: 80371853-80548667 SEMA3C 7.2216 14.9633 -2.07202 0.09333 NM_152754 CHR 7: 84624871-84751247 SEMA3D 3.08308 1.3208 2.33426 0.0246087 NM_012431 CHR 7: 82993221-83278479 SEMA3E 2.09159 2.7487 -1.31417 0.441106 NM_004186 ch r3: 50192847-50226508 SEMA3F 1.39908 3.14351 -2.24683 0.0962234 NM_017789 CHR 2: 97525472-97535735 SEMA4C 1.74128 2.81916 -1.61901 0.153451 NM_020796 CHR 5: 115,779,250 to 115,910,551 SEMA6A 4.71055 3.40428 1.38371 0.140427 NM_0011989 99 ch r15: 47476402-48066420 SEMA6D 4.1238 4.9143 -1.19169 0.55635 NM_153618 chr15: 48010685-48066420 SEMA6D 5.57269 3.1809 1.75192 .1914 NM_025179 CHR 1: 208,195,587 to 208,417,665 PLXNA2 2.30759 2.92134 -1.26597 0.587959

NM_0011055 43 CHR 7: 132,068,246 to 132,261,323 PLXNA4 0.515186 2.28353 -4.43243 0.0424805 NM_020911 CHR 7: 131,808,090 to 132,261,323 PLXNA4 2.00981 4.12258 -2.05123 0.0928033 NM_015103 chr3: 129,274,055 to 129,325,582 PLXND1 2.68071 2.64011 1.01538 0.939199 NM_003873 CHR 10: 33466418-33623833 NRP1 10.2367 15.0383 -1.46906 0.403491 NR_045259 CHR 10: 33466418-33623833 NRP1 10.9158 12.2219 -1.11966 0.814142 NM_201266 ChR2: 206,547,223 to 206,662,857 NRP2 4.96087 4.94254 1.00371 0.99542

The 13 genes whose expression rate was measured on cells before and after implantation in the chicken embryo undergo differential expression variations between these two states.

Analysis of the variations in expression shows that the genes coding for the proteins Sema3C, Sema3F, Sema4C and Plexin A4 have a reduced expression in the cells forming sympathetic tumors, while the expression of the Sema3D and Sema6D genes is markedly increased.

Thanks to this in vivo test, it is shown that these 13 genes are relevant biomarkers whose expression is modulated according to the stage of the tumor and its environment (here, reimplantation of cells in an embryonic environment).

EXAMPLE 4 Inhibition of the Expression of Plexins A1 and A2 in Neuroblastic Cell Lines, Using Interfering RNAs

a) Effects of interfering RNAs directed against plexins on cell proliferation and death by apoptosis

In order to know if the Plexin A1 and / or A2 receptors have an oncogenic role in neuroblastoma, their expression was inhibited by the RNA interference technique, in two types of cell lines: the neuroblastic lines SHEP and IGR-N91 (Figure 4 A).

In the control cells designated by "CTL", a control interfering RNA (siRNA directed against an inert sequence) is introduced by transfection.

Inhibiting the expression of the proteins Plexin A1 (ΡΙχη ou) or A2 (PlxnA2) reduces the proliferation of neuroblastic cancer cells. The effect on cell proliferation is significant, up to -70% during the inhibition of the expression of Plexin A1, and up to more than -30% during the inhibition of the expression of the Plexin A2.

In addition, inhibition of Plexin A1 expression causes significant activation of caspase 3 activity (Figure 4B), and therefore death of cells treated with apoptosis.

This effect is not observed, however, during the inhibition of Plexin A2 expression. It is therefore likely that the two plexins are not involved in the same cellular processes.

b) Inhibition of the expression of plexins A1 or A2 by interfering RNA, on cells in culture in soft agar

A significant decrease in survival and proliferation is observed in the soft agar growth test, resulting in a reduction in cell colonies, both in size and in number (Figura 4C and 4D). The test is carried out on SHEP neuroblastic cells stably expressing GFP.

EXAMPLE 5 Inhibition of the Expression of Plexins A1 and A2 in Neuroblastic Cell Lines, Using Micro-RNAs

By bioinformatics analysis (www.tarqetscan.org), plexins A1 and A2 were identified as potential targets for miR-30 family microRNAs.

The experiment presented is carried out on cells of the HeLa line co-transfected with mir-30 (in polycistronic or mir-30c form alone) and genetic constructs coding for the target genes (ΡΙχηΑΊ, PlxnA2 or Lin28b) integrated into a plasmid miRNAs reporter (pmiR-GIo luciferase reporter System, promega).

FIG. 5 shows that the polycistronic form of the miR-30 microRNA family, like the mir-30c form alone, makes it possible to obtain a significant reduction in the expression of Plexins A1 and A2, by 35% and 45 respectively. %, in vitro.

For comparison, the effects on the Lin28B oncogene, a known target of the mir-30 family (protein expression reduced by 40%) were also measured.

Example 6 Functional Effect of the Subexpression of Sema3C in Neuroblastic Cell Lines

In patient tumor samples, the level of expression of Sema3C is markedly decreased in metastatic tumors of stage 4 or 4S, compared to the level of expression observed in tumors of stage 1,2 or 3 (see Figure 1 B).

In order to determine the effects on cells of this marked under-expression of Sema3C in stage 4 or 4S tumors, a decrease in the expression of a factor 2 of Sema3C was reproduced in cells of neuroblastic line IGR-N91. .

An interfering RNA of sh-RNA type, inducible by doxycycline, was introduced stably into the IGR-N91 cells: FIG. 6A shows the molecular construction carried out.

The transformed cells were grafted into the tissue of a chicken embryo. A tumor thus formed in vivo. Injecting doxycycline into the embryo, halving the expression of Sema3C in the tumor, generated a pro-metastatic signal for this tumor. Tumor cells have detached from the primary tumor tissue and migrated via dissemination routes along the dorsal aorta and peripheral nerves.

Table 3 below summarizes the in vivo effects of this decrease in Sema3C expression:

Table 3. Effects of inducible inhibition of Sema3C on cells

IGR-N91 :: sh-RNA scr. IGR-N91 :: sh-RNA sema3C % of embryos with the following: Control (n = 8) + Doxycycline (N = 6) Control (N = 11) + Doxycycline (N = 12) Implanted tumors in the tissues sympatho- Adrenal 100 100 100 100 Loco dissemination regional 88 67 82 92 Dissemination to distance (metastasis) 38 33 45 92

In control embryos, 33 to 45% of embryos show distant spread from primary tumors. But in the presence of a sh-RNA halving the expression of Sema3C, the rate of embryos presenting a dissemination at a distance from the primary tumor is 92%, that is to say almost all of the embryos which are affected by a metastatic phenomenon. of the primary tumor in vivo.

An ex vivo test was then developed to model the effects induced by the change in expression level of Sema3c on the cohesion of neuroblastoma tumor cells.

The IGR-N91 cells, after transient introduction of siRNA directed against Sema3C, or of a control construction, are cultured in the form of drops, in a culture medium with (on the right) or without Sema3C (on the left of FIG. 6B) .

The percentage of cell aggregation is indicated on the ordinate curve. The black dots represent the percentage of aggregation of cells transformed with an RNA interfere control (siRNA scr). The gray dots represent the percentage of aggregation of cells transformed with an interfering RNA of sh-RNA type directed against the messenger RNA of sema3C (siRNA Sema3c).

In the presence of Sema3c, cells transformed with siRNA Sema3c (gray) regain the aggregation behavior they had lost due to the inhibition of Sema3C expression.

REFERENCES

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WO2016005398

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Claims (13)

1. Method for in vitro molecular characterization of a neuroblastoma tumor, comprising the following steps: a) Measurement in a sample of said tumor of the level of expression of one or more of the 19 genes coding for the proteins chosen from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A , Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2, b) Comparison of the level of expression of said gene (s) measured in step (a) with a reference value determined for the or each of said genes. 2. Method according to claim 1, characterized in that: - In step (a), the expression level is measured for one or more of the 9 genes coding for the following proteins: Sema4C, Sema4D, Sema6D, Plexine A3, Plexine A4, Plexine B2, Plexine C1, Plexine D1 and Neuropiline 1; and - In step (b), for each gene, the reference value is a median of the expression rate of said gene measured in tumors of clinical stage 1,2, 3 and 4S. 3. Method according to claim 1, characterized in that: - In step (a), the expression rate is measured for one or more of the 3 genes coding for the following proteins: Sema4C, Sema3D and Plexin B1; and - In step (b), for each gene, the reference value is a median of the expression rate of said gene measured in tumors of clinical stage 1, 2, 3 and 4. 4. Method according to claim 1, characterized in that: - In step (a), the measurement of the expression rate is carried out for at least the gene coding for the protein Sema3C; and - In step (b), the reference value is a median of the expression rate of said gene measured in tumors of clinical stage 1, 2 and 3. 5. Method according to one of claims 1 to 4, further comprising a step c) of determining the stage of the tumor from one of the stages 1, 2, 3, 4 or 4S, according to the difference observed between the level of expression of at least one gene and its reference value. 6. In vitro method for monitoring the progress of a neuroblastoma tumor in a patient, comprising the following steps: a) Measuring in a sample of said tumor at a time 1Ί the rate of expression of one or more of the 19 genes coding for the proteins as listed in claim 1, b) Comparison of the expression level of said gene or genes measured in step (a) with a reference value determined for the or each of said genes, said reference value determined for each gene having been obtained by measuring the level d expression of said gene in a tumor sample from the same patient at a time T _o , T _o being prior to Ti. 7.. Modulator of the expression or activity of a protein chosen from the group consisting of: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1, Plexin D1, Neuropilin 1 and Neuropilin 2, for use in the treatment of neuroblastoma. 8. A modulator for its use in the treatment of neuroblastoma according to claim 7, characterized in that said modulator is an activator of the expression or activity of Sema3C, and that the neuroblastoma is a neuroblastoma of metastatic stage (4 or 4S). 9. Sema3C for use in the treatment of metastatic stage 4 or 4S neuroblastoma. 10. A modulator for its use in the treatment of neuroblastoma according to claim 7, characterized in that it is an inhibitor of the expression or of the activity of one or more selected plexin (s) ) from Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1 and Plexin D1. 11. Modulator for its use according to claim 10, characterized in that said modulator is an expression inhibitor chosen from an interfering RNA and a micro-RNA, and in particular in that said expression inhibitor is a microRNA of the miR-30 family. 12. Pharmaceutical composition comprising, in a pharmaceutically acceptable medium, at least one therapeutic compound chosen from the group consisting of: a modulator of the expression or of the activity of a protein as defined in one of claims 7, 8, 10 and 11, and - Sema3C. 13. Combination product comprising: at least one therapeutic compound chosen from the group consisting of: a modulator of the expression or of the activity of a protein as defined in one of claims 7, 8, 10 and 11, and Sema3C, and at least a chemotherapy agent or an antiangiogenic agent or an immunotherapy agent.