WO2019030461A1

WO2019030461A1 - Neuroblastoma biomarkers

Info

Publication number: WO2019030461A1
Application number: PCT/FR2018/052052
Authority: WO
Inventors: Valérie Castellani; Céline Delloye-Bourgeois; Arnaud JACQUIER
Original assignee: Universite Claude Bernard Lyon 1; Centre National De La Recherche Scientifique (Cnrs); Institut National De La Sante Et De La Recherche Medicale (Inserm)
Priority date: 2017-08-11
Filing date: 2018-08-09
Publication date: 2019-02-14
Also published as: FR3070047A1

Abstract

The present invention relates to a process for the in vitro molecular characterization of a neuroblastoma tumour, comprising the following steps: a) measuring, in a sample of said tumour, the level of expression of one or more of the 18 genes encoding 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 D1, Neuropilin 1 and Neuropilin 2, b) comparing the level of expression of said gene or genes measured in step (a) with a reference value determined for the or each of said genes, c) determining the stage of the tumour from one of stages 1, 2, 3, 4 or 4S, as a function of the difference observed between the level of expression of at least one gene and the reference value thereof. The present application also relates to a modulator of the expression or of the activity of a protein 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, for use thereof in the treatment of the neuroblastoma.

Description

BIOMARKERS OF NEUROBLASTOMA

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, the growth of cancers comprises two successive stages, firstly the local tumor growth and secondly, the cancerous dissemination.

Local tumor growth consists of the appearance of a tumor clone, from a "transformed" cell, under the action of carcinogenic initiators and promoters. Cell proliferation results in the constitution of the tumor.

Cancerous spread from the initial tumor can be achieved by regional dissemination and / or 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 are derived from the differentiation of neural crest cells during embryogenesis.

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

Tumors from neural crest cells are diverse in nature and include neuroblastoma, melanomas, pheochromocytomas, paragangliomas, chemodectomes, neurogangliomas, PEC (perivascular epithelioid cell) tumors, melanotic neuroectodermal tumors of childhood (MNTI), medullary thyroid carcinomas, gastroenteropancreatic neuroendocrine tumors, sarcoma Ewing and tumors of the same family, malignant tumors of peripheral nerve sheaths (MPNSTs) (also known as "Schwannoma malignant", "Neurofibrosarcoma", and "Neurosarcoma"), neurofibromas, perineuriomas, neurothecomas, nerve sheath myxomas , some type of medulloblastomas, and atypical teratoid and rabdoid tumors.

Among these tumors derived from cells derived from neural ridges, neuroblastoma is a pediatric cancer responsible for more than 15% of cancer-related childhood mortality.

There are several clinical stages of neuroblastoma:

Stages 1, 2 and 3 are characterized by the presence of a more or less voluminous tumor, but strictly localized in the abdomen, sometimes along the vertebral column or in the adrenal glands;

Stage 4 corresponds to the metastatic form, of poor prognosis;

Stage 4S is characterized by the presence of generally important liver and skin metastases, but never bone metastases. This rather particular form regresses in the great majority of the cases, either spontaneously, or following a little aggressive chemotherapy.

Molecular characterization of tumors from embryonic tissues is difficult because there is no healthy tissue corresponding to the tissue of the tumor: indeed, the cells of the neural ridges have disappeared during development, and no longer exist in an organism mammal after birth. Thus, there is no reference tissue with which one can compare the expression levels of genes expressed differently in case of cancer.

Neuroblastomas are highly heterogeneous. Currently, the molecular characterization of neuroblastoma tumors is mainly based on the detection of 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, so for the tumor to progress to stage 4 metastatic. 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 advances, these techniques are not all effective, and 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 intensively treated with chemotherapy, with significant short- and long-term side effects. Only 40% of the 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 for better predicting the evolution of a tumor, in particular to determine its degree of aggressiveness and invasiveness, its probability of relapse after conventional treatment, and new active anti-cancer agents more effective, which act selectively on cancer cells and their signaling pathways, which are less toxic, and which result in fewer, if not no, or very few side effects.

Therapeutic targets specific to cancer cells are therefore actively sought to distinguish between tumors according to their stage, and secondly to identify new treatments targeting different subtypes of neuroblastoma and which are better tolerated by patients.

In order to identify such targets, proteomics studies have shown that some proteins are overexpressed or otherwise under-expressed in neuroblastoma tumors when they become metastatic. The level of expression of these proteins, or their associated genes, is thus 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 thus to adapt the anti-cancer treatment.

Finally, the proteins identified as biomarkers can be used as therapeutic targets. In particular, the proteins overexpressed in the Aggressive metastatic tumors can be advantageously regulated through 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 resulted in encouraging preclinical results.

Overexpression of the anti-apoptosis factor Bcl-2 has been demonstrated in neuroblastic tumors; an inhibition of the expression of this factor results in massive apoptosis of the 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 downregulating the amount of microRNAs in the let-7 family, resulting in a high level of N-MYC protein in neuroblastic cells, inducing the aggressiveness of the tumor. (Molenaar et al., 2012)

Overexpression of Neuropilin 1 was observed in a fast-growing neuroblastoma cell line compared to a cell line of the same tissue origin but at a slower growth rate (Marcus et al., 2005).

Strong expression of Sema3B protein has been reported in benign ganglioneuromas, whereas this protein is virtually undetectable in malignant neuroblastoma tumors (McArdle et al., 2004).

US 2013/0102485 teaches that the presence of miR-9 microRNA is characteristic of stage 4 in neuroblastoma tumors. Under-expression of Plexin C1 protein in stage 4 tumors, compared to the expression seen in stage 1 and 2 tumors, has also been reported.

Other biomarkers and therapeutic targets must now be identified, in order to develop new therapeutic options for patients with neuroblastoma, and to better distinguish different types of tumors, other than by clinical exploration. The object of the present invention is to satisfy these needs. SUMMARY 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, whose expression level, considered for one or more of these proteins, varies according to the stage of the tumor, especially 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 steps of:

a) Measuring in a sample of said tumor the level of expression of one or more of the 18 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 D1, Neuropilin 1 and Neuropilin 2,

b) comparing the level of expression of said gene or genes measured in step (a) with a determined reference value for the or each of said genes,

c) Determination of the stage of the tumor among one of the stages 1, 2, 3, 4 or 4S, as a function of the difference observed between the level of expression of at least one gene and its reference value.

The present invention also relates to a modulator of the expression or activity of at least one protein 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 for its use in the treatment of neuroblastoma.

More particularly, the present invention relates to:

Sema3C for use in the treatment of metastatic stage neuroblastoma (4 or 4S),

an activator of the expression or activity of Sema3C for its use in the treatment of metastatic stage neuroblastoma (4 or 4S); and an inhibitor of the expression or the activity of one or more plexin (s) 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, regardless of its stage. 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 from left to right: stage 1 (st1), stage 2 (st2), stage 3 (st3), stage 4 (st4) and stage 4S (st4S) tumors. Numbers in parentheses indicate the number of samples for each stage. Expression rates are plotted on the ordinate.

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

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

A) Figure 1A. Stage 4 specific biomarkers.

B) Figure 1 B. Biomarker specific for metastatic stages 4 and 4S.

C) Figure 1 C. Biomarkers specific for the 4S stage.

Figure 2. Genetic signature of neuroblastic cell lines SHEP and IGR-N91.

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

Figure 3. Measurement of the expression variation factor of the genes encoding Sema3C, Sema3F and Sema3D proteins, 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 the IGR-N91 cells before they are transplanted into a chicken embryo, and the expression level measured in the IGR-N91 cells. N91 after grafting and incubation for 48 hours of the transplanted embryo. The observed differences are represented as positive values (increase of the level of expression) or negative (decrease of the level of expression) compared to the reference value of the "naive" cells before their transplantation in the embryo.

Figure 4. Effects of 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 lineages SHEP and IGR-N91. Proliferation of cells is measured using the Cell Proliferation Reagent WST-1 kit (Merck).

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

C) Effects of Inhibition of the Expression of Plexin A1 (PIxnAI) and Plexin A2 (PlxnA2) Using Interfering RNAs (siPLxnAI and siPlxnA2) in a Soft Agar Growth Test on SHEP Neuroblastic Cells , using as control an interfering RNA directed against an inert sequence (siCTL). The clones are visualized using fluorescence emitted by GFP, stably expressed in SHEP cells.

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

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

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

Figure 6. Pro-metastatic role of Sema3C

A) Genetic construct for inducing, in the presence of doxycycline, the expression of a sh-RNA directed against Sema3C messenger RNA. The corresponding results are shown in Table 3. B) The IGR-N91 cells, after transient introduction of siRNA directed against Sema3C, or of a control construct, are cultured in 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 Sema3C protein. The percentage of cellular 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 cells transformed with sh-RNA-like interfering RNA directed against the sema3C messenger RNA (siRNA Sema3c).

DETAILED DESCRIPTION OF THE INVENTION

Preliminary remarks

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

The Semaphorin / Neuropiline / Plexin signaling pathway consists of a network of functionally associated genes, encoding secreted and / or transmembrane receptors (neuropilins and plexines) 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 comprises 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 approximately 500 amino acids, located near the N-terminus, essential for the activity of these proteins. Semaphorin class 3 (Sema 3A, 3B, 3C, 3D, 3E and 3F) are the only ones to be secreted and non-transmembrane in vertebrates.

The family of plexines includes nine transmembrane receptors, characterized by the presence in their extracellular domain of a "sema" type domain of about 500 amino acids, similar to that present in the proteins of the semaphorin family. Plexins are the receptors of semaphorins, in some cases in combination with neuropilins, and transmit their signal through the activation of pathways intracellular. The plexins all have, in their intracellular domain, a GAP (GTPase-activating protein) domain separated in two by a RBD domain (RHO-GTPase-binding domain). Dimerization of the plexines would activate the GAP domain.

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

Each plexin has a specificity for binding one or more semaphorins. For example, plexin A is the receptor for semaphorin 6: plexin A1 specifically binds Sema6D, while plexin A2 and A4 interchangeably bind semaphorin Sema6A and Sema6B. All A plexins 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 especially in processes such as angiogenesis, heart development, skeletal morphogenesis and morphogenesis kidney. (For review, see Peràlà et al., 2012).

NeuropilineV and Neuropiline2 receptors (NPR1, NPR2) act as semaphorin co-receptors, in association with the plexines that transmit the signal to the cell by activating their GAP domain.

Studies on the functional involvement of this signaling pathway, in the context of nervous system development, show that interactions between members of this pathway regulate cell behavior. Thus, the coexpression 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 Sema3A / Neuropiline1 pair 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 instead reinforces the response to the third (Sanyas et al, 2012). In general, there is a control of the level of expression of neuropilins on the cell surface, which determines the sensitivity of the cells to the ligands. In addition, the co-expression of plexin A and their transmembrane semaphorin ligands inhibits the cellular response to exogenous semaphorins. Finally, it has also been shown that, depending on the members of the pathway expressed, and its signaling effectors within the cell, the final functional effect may be a promoter or promoter effect. on the contrary inhibits 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, have recently been described in the genome of neuroblastoma tumors (Li et al., 2017).

Molecular characterization method of a neuroblastoma tumor

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

a) measuring in a sample of said tumor the level of expression of one or more of the genes encoding the proteins selected from the group consisting of: a semaphorin, a plexin and a neuropiline,

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

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

a) Measuring in a sample of said tumor the level of expression of one or more of the 18 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 D1, Neuropilin 1 and Neuropilin 2,

c) Determination of the stage of the tumor among one of the stages 1, 2, 3, 4 or 4S, as a function of the difference observed between the level of expression of at least one gene and its reference value. For the purposes of the invention, a method of molecular characterization denotes an in vitro method making it possible to obtain various information on the tumor: stage, risk of spread in the body, aggressiveness, resistance to certain therapeutic treatments, etc. characterization is independent of clinical examination.

Tumor samples are obtained according to the usual techniques, by qualified experimenters. In particular, samples from patients can be made 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 level of expression 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 cells.

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

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

a) Measuring in a sample of said tumor the rate of production of one or more of the 18 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 D1, Neuropilin 1 and Neuropilin 2,

b) Comparison of the production rate of the protein or proteins measured in step (a) with a determined reference value for the or each of said proteins, c) Determination of the stage of the tumor among one of stages 1, 2 , 3, 4 or 4S, depending on the observed difference between the level of expression of at least one gene and its reference value.

This method of characterization may be carried out in place of that comprising the measurement of the level of expression of genes, or be carried out in addition to the method comprising measuring the level of expression of genes. Moreover, within the meaning of the invention, the measurement of the rate of production of one or more proteins includes the measurement of the level of surface presence of the membrane-bound or membrane-associated proteins, and / or the level of secretion of the proteins by the cell.

In particular, the level of surface presence of membrane or membrane-associated proteins, and the amount of proteins secreted by the cell, can be compared which will give additional indications on the molecular characterization of the tumor.

The term "reference value" refers to the numerical value of a level of expression of a gene in one or more tumors whose stage (s) is (are) known. For a gene (i), this value will be referred to below as Vi.

According to a preferred aspect of the invention, the reference value Vi is a mean or a median of several values of the expression level of the same gene, measured in various known stage tumors.

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 4 tumors. Other reference values can be defined, as will be presented later.

The comparison of the value obtained in step (a) 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 indistinctly, and both refer to either a significant increase or a significant decrease in the value of the expression level of a Ti gene relative to its reference value Vi.

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

According to one embodiment of the invention, the measured reference values and expression levels can be normalized to take into account the amount of cellular biomass. This normalization of the relative values obtained for each gene is obtained by dividing these values by the value of the expression level 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 HPRT protein (hypoxanthine-guanine phosphoribosyltransferase). This same standardization technique of the relative values can be implemented when the measured rates are protein production rates.

The method further comprises a step c) of determining the stage of the tumor among one of stages 1, 2, 3, 4 or 4S, depending on the difference observed between the expression level Ti of at least one gene as cited above, and its reference value Vi.

The term "stage determination" refers to a particular aspect of the molecular characterization of the tumor by assigning 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 implements a technology that is independent of conventional clinical investigations.

Advantageously, the method according to the invention can be carried out by measuring, in the tumor sample, the expression level 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 D1, Neuropilin 1 and Neuropilin 2.

Indeed, each biomarker can be used independently of the other biomarkers to characterize in a molecular way the tumor from which the studied sample originates.

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

Such molecular characterization of a tumor allows the investigators to determine, as a function of the differences observed with the reference values Vi for each gene (i), as accurately as possible the "status" of the tumor, this including the stage, the risk of metastasis, aggression, invasiveness and the chances of response to treatment of said tumor. For tumors of the same characteristic, the variations of this signature are representative of differences between the patients. Advantageously, the molecular characterization of a tumor according to the method of the invention may allow the health care staff to choose the most appropriate therapeutic treatment for the tumor thus characterized, and thus to practice a personalized medicine.

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

Among all these combinations of 18 genes, it will be possible in particular 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 neuropiline;

at least one semaphorin and at least one plexin;

at least one semaphorin and at least one neuropiline;

at least one semaphorin;

at least Sema3C;

at least two semaphorins and at least two plexines;

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

at least three semaphorins, at least four plexines and at least one neuropiline;

at least Sema4C, Sema4D, Sema6D, Plexin A3, Plexin A4, Plexin B2, Plexin D1 and Neuropilin 1.

It is understood that, according to the method of the invention, the expression levels of other genes than those mentioned above can be measured, and the values obtained can be used to determine the stage of the tumor. In particular, the level of expression of the gene coding for Plexin C1 may be measured, in addition to the measures previously described. According to a first implementation of the invention, in step (a), the measurement of the level of expression is carried out for one or more of the 8 genes encoding the following proteins: Sema4C, Sema4D, Sema6D, Plexin A3, Plexin A4, Plexin B2, Plexin D1 and Neuropilin 1.

Preferably, the level of expression of the gene coding for Plexin C1 may also be measured during this implementation.

This particular combination of nine biomarkers is particularly suitable for distinguishing stage 4 tumors, as shown in Figure 1A. Indeed, all these genes have a level of expression significantly different from those found in samples of stage 1, 2, 3 or 4S tumors.

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

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

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

In step (a), the measurement of the level of expression is carried out for one or more of the 8 genes encoding the following proteins: Sema4C,

Sema4D, Sema6D, Plexin A3, Plexin A4, Plexin B2, Plexin D1 and Neuropilin 1, and

In step (b), for each gene, the reference value is a median of the expression level of said gene measured on 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) Measuring in a sample of said tumor the level of expression of one or more of the 8 genes encoding the proteins selected from the group consisting of: Sema4C, Sema4D, Sema6D, Plexin A3, Plexin A4, Plexin B2, Plexin D1 and Neuropilin 1, b) comparing the level of expression of said gene or genes measured in step (a) with a determined reference value for the or each of said genes, this reference value being a median of the expression level of said gene measured on clinical stage 1, 2, 3 and 4S tumors, and

c) Determination of the stage of the tumor as a function of the observed difference between the level of expression of at least one gene and its reference value.

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

According to a second implementation of the invention, in step (a), the measurement of the level of expression is carried out for one or more of the 3 genes encoding the following proteins: Sema4C, Sema3D and Plexin B1.

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

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

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

In step (a), the measurement of the level of expression is carried out 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 on 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) measuring in a sample of said tumor the level of expression of one or more of the 3 genes encoding the proteins selected from the group consisting of: Sema4C, Sema3D and Plexin B1,

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

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

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

The use of this biomarker is particularly suitable for distinguishing between stage 4 or 4S tumors, ie metastatic tumors, as shown in figure 1 B. In fact, this gene has a high of significantly different expression (its expression is markedly diminished) from those found in samples of stage 1, 2, and 3 tumors.

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

The reference value V may in particular be the result of the arrhythmic average, or the median, calculated from the expression levels obtained on tumors whose stage 1, 2, 3 is known.

According to a specific implementation, the present invention relates to a method for in vitro characterization of a neuroblastoma tumor, in which: In step (a), the measurement of the level of expression is carried out for at least the gene encoding the Sema3C protein; and

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

a) measuring in a sample of said tumor the level of expression of at least the gene encoding the Sema3C protein,

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

c) Determination of the stage of the tumor as a function of the difference observed between the level of expression of at least this gene and its reference value.

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

Monitoring method of a neuroblastoma tumor

The method according to the present invention advantageously allows to follow tumor revolution in a given patient.

For this, the method of in vitro molecular characterization of a tumor will be performed on samples from the same tumor, but obtained at different times, especially before and after the start of a therapeutic treatment, or before and after an intervention. surgical procedure, or after a remission and then during a recurrence of the tumor.

More generally, the preliminary step of obtaining the sample of the tumor may be carried out at a time Ti, and the reference value will be that obtained by measurement the level of expression of a gene on a tumor sample obtained at a time T ₀ , T ₀ being prior to Ti.

Thus, the invention relates to an in vitro method for monitoring the evolution of a neuroblastoma tumor in a patient, comprising the following steps:

a) measuring in a sample of said tumor at a time T-1 the expression rate of one or more of the 18 genes encoding the proteins as listed above,

b) comparing the expression level of said gene or genes measured in step (a) with a determined reference value for the or each of said genes, said reference value determined for each gene having been obtained by measuring the rate of expression of said gene in a tumor sample of the same patient at a time T ₀ , T ₀ being prior to T

According to a particular implementation of the method, the time Ti is located chronologically after the beginning of the administration of a therapeutic treatment to the patient; and the time T ₀ is located chronologically before the beginning of said treatment.

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

According to this aspect, the present invention relates to an in vitro method of monitoring the evolution of a neuroblastoma tumor in a patient, comprising the following steps:

a) Measuring in a sample of said tumor the level of expression of one or more of the 18 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,

A4 Plexin, Plexin B1, Plexin D1, Neuropilin 1 and Neuropilin 2,

b) comparing the expression level of said gene or genes measured in step (a) with a determined reference value for the or each of said genes, said reference value determined for each gene having been obtained by measuring the rate of expressing said gene in a sample of said tumor of the same patient obtained at a time T ₀ , T ₀ being earlier than Ti. DNA chips and diagnostic kits

The invention also relates to a DNA chip carrying at least 18 oligonucleotide probes, each of the probes being specific for a gene among the genes encoding the following proteins: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin BI, Plexin D1, Neuropilin 1 and Neuropilin 2.

According to one particular embodiment, said DNA chip will bear at least the 18 oligonucleotide probes specific for the genes coding for the proteins mentioned above.

According to one particular embodiment, said DNA chip will carry other oligonucleotide probes in addition to the abovementioned 18, and in particular may carry a probe specific for the gene coding for the Plexin C1 protein.

The present invention also relates to a diagnostic kit for implementing 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 encoding the following proteins: Sema3C, Sema3A, Sema3B, Sema3D, Sema3E, Sema3F, Sema4C, Sema4D, Sema6A, Sema6D, Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin D1, Neuropilin 1 and Neuropilin 2.

The invention also relates to the use of this DNA chip and / or 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 evolution of a neuroblastoma tumor in a patient.

Therapeutic compounds for use in the treatment of neuroblastoma

The term "treating cancer" is intended to slow down or completely inhibit the development of a tumor in the patient, and / or to remove completely from the body of the patient all the cancer cells and in particular the tumor of the patient. 'origin. More specifically, the concept of "treatment of neuroblastoma" covers several aspects such as the reduction of the tumor size, the disappearance of the primary tumor and / or secondary tumors (metastases) and the prevention of recurrence.

Pharmaceutical compositions intended to treat certain cancers, including semaphorins, have previously been described (see in particular the international application WO 2009/056091). However, none of these compositions have been contemplated to specifically treat neuroblastoma.

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

By therapeutic compound is meant a compound effective to slow or inhibit the development of a neuroblastoma tumor in the patient, and / or a compound for removing a neuroblastoma tumor. By "tumor" is meant a primary tumor or a secondary tumor (metastasis) resulting from the dissemination of the primary tumor.

More specifically, within the meaning of the invention, such a therapeutic compound consists of a modulator of the expression or activity of at least one protein 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.

The invention therefore relates to a modulator of the expression or activity of at least one protein 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 for its use in the treatment of neuroblastoma.

According to one particular embodiment of the invention, the expression or activity modulator is that of at least one protein 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 D1, Neuropilin 1 and Neuropilin 2. The term "expression or activity modulator" refers to a compound capable of specifically influencing the expression or activity of a protein selected from the list above.

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

Those skilled in the art will be able to choose among the different compounds that may have the required activity: for example, the inhibition of the expression of a protein may be carried out using interfering RNA or microRNAs; the inhibition of its activity can be achieved by the use of antagonists, especially antibodies; and the stimulation of its expression can be obtained by means of stabilizing compounds of the 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 inhibitory or expression-activating compounds. the activity of said proteins.

The present invention also relates to a method for treating neuroblastoma, comprising administering to a patient afflicted with this cancer a modulator of the expression or activity of at least one protein 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.

According to one particular embodiment, said modulator is an activator of Sema3C expression or activity, and the neuroblastoma treated is a metastatic stage neuroblastoma (4 or 4S).

The invention also relates to the Sema3C protein for its use in the treatment of metastatic stage neuroblastoma (4 or 4S).

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 case of a decrease in the expression of Sema3C, the cells that 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 its activity, or any compound capable of mimicking its activity, is likely to be used in the treatment of metastatic stage neuroblastoma.

It is understood that the term "Sema3C" refers to both the protein as such, but also its derived forms as well as peptides or agonists mimicking the activity of this protein, and also includes nucleic acids encoding a protein Sema3C, human or not, or their homologues encoding a protein of another species, having the same biological function.

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

According to one particular embodiment, said modulator for its use in the treatment of neuroblastoma is an inhibitor of the expression or 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 one particular embodiment, said modulator is an inhibitor of the expression or the activity of at least one plexin chosen from Plexin A1 and Plexin A2.

More particularly, said modulator is an inhibitor of the expression or activity of Plexin A1.

More particularly, said modulator is an inhibitor of the expression or activity of Plexin A2.

The present invention also relates to a method for treating neuroblastoma, comprising administering to a patient suffering from this cancer an inhibitor of the expression or activity of at least one plexin selected 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 acting on one of the activities below, by annoying it:

(i) the ability of plexin to form a homodimer, (ii) its ability to bind its natural ligand (s),

(iii) its ability 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 include 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 semaphorin, will be in particular 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 hindering the semaphorin binding site.

An inhibitor acting on one of the plexin-activated intracellular signaling pathways will notably 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 to block the intracellular protein binding site 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 its ability to form a homodimer and bind its natural ligand.

According to one particular embodiment 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.

The person skilled in the art knows many ways of reducing the expression of a protein, which include in a nonlimiting manner all the genetic modification technologies, and all the factors that inhibit the transcription of the gene coding for a plexin, or else the translation. messenger RNA to the protein; these means include the use of interfering RNAs and microRNAs.

Such an inhibitor of plexin expression will in particular be an effectively reducing inhibitor, that is to say at least 40%, more preferably at least 60%, or even better at least 80% , the expression of the plexin against which it is directed. More particularly, the inhibitor of the expression or activity of at least one plexin may be an expression inhibitor selected from an interfering RNA and a microRNA.

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

The interfering RNAs are RNAs hybridizing to the messenger RNA (mRNA) gene expression product 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 more than 99% inhibition.

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

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

SiRNAs can also be ordered from specialized trading companies, such as Santa Cruz and Sigma-Aldrich, which provide pre-designed siRNAs upon request.

Among the interfering RNAs, there will also be mentioned "shRNA" or small hairpin RNA, which are RNAs adopting a stem and loop structure.

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

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

Interfering RNAs capable of inhibiting the expression of certain plexins have been described in the prior art, and are incorporated herein by reference.

We can mention in particular:

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

shRNA interfering RNAs inhibiting the expression of A1, A2, A3 and A4 plexines were used in HUVEC endothelial cells (Kigel et al., 201 1);

interfering shRNA-like RNAs inhibiting plexin A1 expression were introduced into MGC803 gastric cancer cells, resulting in decreased levels of VEGFR2 (Lu et al., 2016);

interfering shRNA-like RNAs inhibiting plexin A1 expression were used in lung carcinoma cells (Yamada et al., 2016).

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

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

The 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 the translational repression or to the degradation of this messenger RNA.

In humans, microRNAs are abundant in a large number of cell types, and would target about 60% of genes. Thus, therapies based on miRNAs are currently under study, these therapies aiming to specifically inhibit the expression of certain genes, notably 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 microRNA 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. Expression of the miR-30 family is greatly diminished in aggressive neuroblastomas, suggesting that these may regulate tumor growth of neuroblastomas.

The results of the experiments conducted by the inventors, presented in FIG. 5, demonstrate that this microRNA has a definite effect on the expression of A1 and A2 plexines, the expression inhibition being significant and being accompanied by a functional effect since the Neuroblastoma cells exhibit diminished proliferation when plexin expression is thereby inhibited.

The present invention also relates to a vector for the expression of a microRNA 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 expression of said microRNA in a host cell.

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

The present invention also relates to a pharmaceutical composition for its use in the treatment of neuroblastoma, comprising in a pharmaceutically acceptable medium, at least one therapeutic compound selected from the group consisting of:

a modulator of the expression or activity of at least one protein 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,

an activator of the expression or activity of Sema3C, - Sema3C, and

an inhibitor of the expression or activity of at least one plexin selected from Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin

C1 and Plexin D1.

A pharmaceutically acceptable medium denotes a vehicle for preserving and administering the inhibitor according to the invention, and optionally excipients, whose administration 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 for its use according to the invention may comprise any necessary pharmaceutical excipient, such as buffering agents or agents for adjusting pH or isotonicity, or wetting agents. A pharmaceutical composition according to the invention may also comprise one or more antioxidant agents, and / or one or more preserving agents.

A pharmaceutical composition as described above may be administered by any suitable route, such as orally, buccally, sublingually, ophthalmically, rectally, topically, or parenterally, in particular by an intraperitoneal, intradermal, subcutaneous route. , intravenous or intramuscular.

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

According to another embodiment, a pharmaceutical composition for its use according to the invention may be prepared for parenteral administration, in 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 for its use according to 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 may be used, for example water, saline buffer solution, 0.4% or 0.3% glycine solution, or a solution of hyaluronic acid.

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

According to a preferred embodiment 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 against cancer cells. The effective amount of such an inhibitor may depend on a parameter or a plurality of parameters, such as the route of administration, single or multiple dose administration, patient characteristics, including age. physical condition, height, weight and 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 a therapeutic compound, an interfering RNA or a microRNA which inhibits 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 for use in the treatment of neuroblastoma comprising:

at least one therapeutic compound selected from the group consisting of: a modulator of the expression or activity of at least one protein 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,

o an activator of the expression or activity of Sema3C,

o Sema3C, and

an inhibitor of the expression or the 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,

and

at least one chemotherapeutic agent or an anti-angiogenic agent or an immunotherapeutic agent.

A chemotherapeutic agent that is suitable for the invention may be selected from cytostatic agents, antimetabolites, DNA intercalators, topoisomerase I and II inhibiting compounds, tubulin inhibitors, alkylating agents, neocarcinostatin, calichéamicine. , dynemicine or hopeamicin A, compounds inhibitors of ribosomes, tyrosine phosphokinase inhibitors, cell differentiation inducing compounds, or histone deacetylase inhibitors.

In particular, a chemotherapy agent that is suitable for the invention may be chosen, in a nonlimiting manner:

· From cytostatics and antimetabolites, such as 5-fluorouracil, 5-fluoro-cytidine, 5-fluoro-uridine, cytosine arabinoside or methotrexate,

• among DNA intercalators, such as doxorubicin, daunomycin, idarubicin, epirubicin or mitoxantrone, · among topoisomerase I and II inhibiting compounds, such as camptothecin, etoposide or m-AMSA,

• among tubulin inhibitors, such as vincristine, vinblastine, vindesine, taxol, nocodazole or colchicine,

Among alkylating agents, such as cyclophosphamide, mitomycin C, rachelmycin, cisplatin, mustard phosphoramide gas, melphalan, bleomycin, N-bis (2-chloroethyl) -4-hydroxyaniline,

• among neocarcinostatin, calichéamicine, dynémicine or espéramicine A,

Ribosomal inhibiting compounds, such as verrucarin A, among tyrosine phosphokinase inhibitors, such as quercetin, genistein, erbstatin, tyrphostin or rohitukin and its derivatives,

Among compounds inducing cell differentiation, such as retinoic acid, butyric acid, phorbol esters or aclacinomycin, or

· Among histone deacetylase inhibitors, such as CI-994 or MS275.

An angiogenesis inhibiting agent that is suitable for the invention may be chosen from the following nonlimiting lists:

monoclonal antibodies targeting VEGF such as Bevacizumab, protein compounds mimicking VEGF receptor domains, such as Aflibercept,

inhibitors of tyrosine phosphokinases such as Sorafenib,

Sunitinib, Pazopanib, Axitinib, Vantetanib or Cabozantinib, rapamycin analogues such as Temsirolimus or Everolimus. An immunotherapy agent suitable for the invention may be chosen from cytokines, such as interleukin-2,

cells of the immune system, including immune cells of the patient removed, activated and reinjected to 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 a radiotherapy treatment.

A radiotherapy treatment that 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-borne 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 provenance

Statistical analysis of the data was performed with the Prism 6.0e software (GraphPad).

Example 1. Gene Expression Patterns of Semaphorin Signaling Pathway in Neuroblastoma Tumor Specimens at Different Stages

The cohorts of patients studied are presented in the following bibliographical references:

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

- Shi & Fisher GSE49710 Cohort: Wang et al., 2014; this cohort groups together 498 tumor samples.

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

a) Stage 4 biomarkers

Figure 1A shows the expression level measurements performed on neuroblastic tumor samples from the Wolf & Kocak cohort of GSE45480 patients.

One or two probes were used for each gene.

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

Nine genes showed 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 the D1 and B2 plexines are not linked to a poor survival prognosis. b) Biomarkers of metastatic stages 4 and 4S

Figure 1B shows the expression rate measurement of the gene encoding Sema3C performed on neuroblastic tumor samples from the cohort of Kocak patients (GSE45480).

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

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

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

c) Specific stage 4S biomarkers

Figure 1C shows the expression level measurements performed on neuroblastic tumor samples 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 4S stage tumor samples, relative to the levels of expression seen in stage 1, 2, 3 and 4 tumors.

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 expression level of seven genes was measured in a panel of 26 primary lines and 4 immortalized lines from stage 4 neuroblastoma tumors. Expression quantified by quantitative RT-PCR (RT-qPCR) reveals differences expression rate for each gene, from one line to another. (Results not shown) b) The expression rate of nine genes was measured in immortalized cells derived from neurobastoma, the IGR-N91 and SHEP lines. The technique used is RT-qPCR, the amplified nucleic acid level being normalized to that of the gene encoding the HPRT household protein. Figure 2 presents 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 level of expression of these genes presented here does not allow 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 in terms of a diagnosis or follow-up procedure.

Example 3. Semaphorin Signaling Pathway Gene Expression Patterns in Stage 4 Neuroblastoma Tumor Specimens, After Transplantation into HH14 Chicken Embryos and 48 Hour Incubation

Neuroblastoma tumors were produced in vivo by micrografting human tumor cells in the tissues of an avian embryo, a tumorignin model set forth in patent application WO 2016/005398.

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

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

This analysis revealed a signature of expression modifications of 13 genes of the Semaphorin / Neuropiline / Plexin network, which correlates the analysis data of the expressions in the cohorts of patients.

a) Cells of the neuroblastic cell line IGR-N91 were grafted into a chicken embryo according to the protocol set forth 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 encoding Sema3C, Sema3F and Sema3D were determined by quantitative RT-PCR, both on the IGR-N91 cells before the grafting ("naive cells"), and on the tumors taken from the Chicken embryo 48 after this transplant.

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

b) Cells derived from neuroblastoma tumors were grafted into a chicken embryo according to the protocol set forth 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 the tumor cells before the transplantation ("naive cells"), and on the tumors taken from the chicken embryo. 48 after this transplant.

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

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

Number Chromosomal Locus Gene Mean Average Variation p-value of accession coding rate rate between (eqvar)

(platform for expresso expressio two

NCBI) protein: n in ion in types of

tumors the cells

taken "naive

cells »

NM_006080 chr7: 83587658-83824217 SE M A3 A 8,53902 6.96972 1, 22516 0.439958

NM_006379 chr7: 80371853-80548667 SEMA3C 7.2216 14.9633 -2.07202 0.09333

NM_152754 chr7: 84624871 -84751247 SEMA3D 3.08308 1, 3208 2.33426 0.0246087

NM_012431 chr7: 82993221 -83278479 SEMA3E 2.09159 2.7487 -1, 31417 0.441106

NM_004186 chr3: 50192847-50226508 SEMA3F 1, 39908 3.1435 -2.24683 0.0962234

NM_017789 chr2: 97525472 -97535735 SEMA4C 1, 74128 2.81916 -1, 61901 0.153451

NM_020796 chr5: 115779250-115910551 SEMA6A 4.71055 3.40428 1.38371 0.140427

NM_001 1989 chrl 5: 47476402? 8066420 SEMA6D 4.1238 4.9143 -1, 19169 0.55635

99 NMJ 53618 chrl 5: 48010685? 8066420 SEMA6D 5.57269 3.1809 1, 75192 0.1914

NM_025179 chr1: 208195587-208417665 PLXNA2 2.30759 2.92134 -1, 26597 0.587959

NM_001 1055 chr7: 132068246-132261323 PLXNA4 0.515186 2.28353 -4.43243 0.0424805 43

NM_020911 chr7: 131808090-132261323 PLXNA4 2.00981 4.12258 -2.05123 0.0928033

NM_015103 ch r3: 129274055-129325582 PLXND1 2.68071 2.64011 1, 01538 0.939199

NM_003873 chrl 0: 33466418-33623833 NRP1 10.2367 15.0383 -1, 46906 0.403491

NR_045259 chrl 0: 33466418-33623833 NRP1 10.9158 12.2219 -1, 1 1966 0.814142

NM_201266 chr2: 206547223-206662857 NRP2 4.96087 4.94254 1, 00371 0.99542

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

Expression variation analysis shows that the genes encoding the Sema3C, Sema3F, Sema4C and A4 Plexin proteins have reduced expression in sympathetic tumor-forming cells, while the expression of 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 the cells in an embryonic environment).

EXAMPLE 4 Inhibition of A1 and A2 Plexin Expression in Neuroblastic Cell Lines Using Interfering RNAs

a) Interfering RNA effects against plexines on cell proliferation and apoptosis death

In order to know whether 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 lineages SHEP and IGR-N91 (FIG. 4 A). In control cells designated 'CTL', a control interfering RNA (siRNA directed against an inert sequence) is introduced by transfection. Inhibition of Plexin A1 (PIxnAI) or A2 (PlxnA2) protein expression reduces the proliferation of neuroblastic cancer cells. The effect on cell proliferation is important, up to -70% when inhibiting the expression of Plexin A1, and up to more than -30% when inhibiting the expression of Plexin A1. Plexin A2.

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

This effect is however not observed when inhibiting the expression of Plexin A2. It is therefore likely that the two plexines are not involved in the same cellular processes.

b) Inhibition of the expression of A1 or A2 plexines by interfering RNAs on cells in soft agar culture

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 number (Figure 4C and 4D). The test is performed on SHEP neuroblastic cells stably expressing GFP.

Example 5 Inhibition of A1 and A2 Plexin Expression in Neuroblastic Cell Lines Using Micro-RNAs

Through bioinformatic analysis (www.targetscan.org), A1 and A2 plexines have been identified as potential targets for microRNAs in the miR-30 family.

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

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

For comparison, the effects on the Lin28B oncogene, a known target of the mir-30 family (reduced protein expression by 40%) were also measured. Example 6. Functional Effect of Sema3C Under-expression in Neuroblastic Cell Lines

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

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

A doxycycline-inducible sh-RNA-like interfering RNA was stably introduced into IGR-N91 cells: Figure 6A shows the molecular construction performed.

Cells thus transformed were grafted into the tissues of a chicken embryo. A tumor was thus formed in vivo. The injection of doxycycline into the embryo, halving the expression of Sema3C in the tumor, generated a pro-metastatic signal for this tumor. Tumor cells disassociated from primary tumor tissue and migrated through dissemination pathways 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

% Control embryos (n = 8) + Doxycycline Control + Doxycycline with the following elements (n = 6) (n = 1 1) (n = 12):

Implanted tumors 100 100 100 100

in the tissues

sympatho- Adrenal

Local release 88 67 82 92

regional

Dissemination at 38 33 45 92

distance (metastases) In control embryos, 33 to 45% of embryos have a distant spread of primary tumors. But in the presence of a sh-RNA halving the expression of Sema3C, the rate of embryos with distant dissemination of the primary tumor is 92%, almost all the embryos that are affected by a metastatic phenomenon. of the primary tumor in vivo.

An ex vivo assay was then developed to model the effects induced by Sema3c expression level change on neuroblastoma tumor cell cohesion.

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

The percentage of cellular 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 cells transformed with sh-RNA-like interfering RNA directed against the sema3C messenger RNA (siRNA Sema3c).

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

REFERENCES

PATENTS

WO 2016/005398

US 2013/0102485

WO 2009/050691

NO PATENTS

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Claims

A method of in vitro molecular characterization of a neuroblastoma tumor, comprising the steps of:

Method according to claim 1, characterized in that:

In step (a), the measurement of the level of expression is carried out for one or more of the 8 genes encoding the following proteins: Sema4C, Sema4D, Sema6D, Plexin A3, Plexin A4, Plexin B2, Plexin D1 and Neuropiline 1; and

Method according to claim 1, characterized in that:

In step (a), the measurement of the level of expression is carried out for one or more of the 3 genes encoding 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 on clinical stage 1 tumors,

2

3 and 4.

4. Method according to claim 1, characterized in that:

In step (a), the measurement of the level of expression is carried out for at least the gene encoding the Sema3C protein; and

An in vitro method of monitoring the evolution of a neuroblastoma tumor in a patient, comprising the steps of:

a) measuring in a sample of said tumor at a time T-1 the expression rate of one or more of the 18 genes encoding the proteins as listed in claim 1,

said reference value determined for each gene having been obtained by measuring the level of expression of said gene in a tumor sample of the same patient at a time T ₀ , T ₀ being prior to T

A modulator of the expression or activity of at least one protein 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, for its use in the treatment of neuroblastoma.

Modulator for its use in the treatment of neuroblastoma according to claim 6, characterized in that said modulator is an activator of the expression or activity of Sema3C, and that the neuroblastoma is a metastatic stage neuroblastoma (4 or 4S).

8. Sema3C for use in the treatment of metastatic stage neuroblastoma (4 or 4S).

9. Modulator for use in the treatment of neuroblastoma according to claim 6, characterized in that it is an inhibitor of the expression or the activity of one or more plexin (s) chosen (s) ) among Plexin A1, Plexin A2, Plexin A3, Plexin A4, Plexin B1, Plexin C1 and Plexin D1.

10. Modulator for use according to claim 9, characterized in that said modulator is an expression inhibitor selected from an interfering RNA and a microRNA, and in particular that said expression inhibitor is a microRNA of the miR-30 family.

1 1. A pharmaceutical composition for use in the treatment of neuroblastoma comprising, in a pharmaceutically acceptable medium, at least one therapeutic compound selected from the group consisting of:

a modulator of the expression or activity of a protein as defined in one of claims 6, 7, 9 and 10, and

- Sema3C.

12. Combination product for use in the treatment of neuroblastoma comprising:

at least one therapeutic compound selected from the group consisting of: a modulator of the expression or activity of a protein as defined in one of claims 6, 7, 9 and 10 and Sema3C, and at least one chemotherapeutic agent or an anti-angiogenic agent or an immunotherapy agent.