ACKR4 as a biomarker for the prognosis of b-cell non-hodgkin lymphoma

Abstract

The present invention relates to Ackr4 as a biomarker for prognosing clinical outcome in a male subject suffering from a B-Cell Non-Hodgkin Lymphoma. It also relates to methods for predicting clinical outcome and methods for monitoring the effect of a therapy or the progression of a B-Cell Non-Hodgkin lymphoma. Another aspect of the invention relates to the ACKR4 protein for use in the treatment or the prevention of a B-Cell Non-Hodgkin Lymphoma.

Description

[0001] ACKR4 AS A BIOMARKER FOR THE PROGNOSIS OF B-CELL NON-HODGKIN LYMPHOMA

[0002] The present invention relates to Ackr4 as a biomarker for prognosing clinical outcome in a male subject suffering from a B-Cell Non-Hodgkin Lymphoma. It also relates to methods for predicting clinical outcome and methods for monitoring the effect of a therapy or the progression of a B-Cell Non-Hodgkin lymphoma. Another aspect of the invention relates to the ACKR4 protein for use in the treatment or the prevention of a B-Cell Non-Hodgkin Lymphoma.

[0003] Burkitt's lymphoma is a very rapidly growing non-Hodgkin's B-cell lymphoma. It is a MYC-driven lymphoma which comprises three clinical subtypes: an endemic variant that occurs in equatorial Africa and is associated with Epstein-Barr virus (EBV) in almost all cases, a sporadic variant that occurs throughout the world and an immunodeficiency-related variant that is most common in people with acquired immunodeficiency syndrome (AIDS) or with immune deficiencies. Its incidence rate is more than twice higher in males than in females regardless of age.

[0004] Burkitt's lymphoma presents with rapidly enlarging lymph node tumor masses involving the chest and / or the abdomen. It has a high tendency to spread to the central nervous system (brain and spinal cord), and can also involve the liver, spleen and bone marrow.

[0005] As Burkitt lymphoma is fast growing, its treatment must be initiated quickly after diagnosis and can be very intensive lasting several months. It is usually treated with a number of different chemotherapy drugs and a targeted immunotherapy. With an intense treatment, disease prognosis is good in children but poor in adults.

[0006] In this context, there remains a need for new tools for improving the early prognosis and management of patients suffering from Burkitt's lymphoma.

[0007] The present invention is believed to meet such a need by providing a new biomarker to predict clinical outcome and to assess chemotherapy treatment responsiveness in Burkitt's lymphoma patients.

[0008] The ACKR4 gene encodes an atypical chemokine receptor. By reducing the availability of chemokines such as CCL19, CCL21 and CCL25, ACKR4 plays an important role in controlling migration of immune or cancer cells that express CCR7 or CCR9 chemokine receptors. There is also increasing evidence that ACKR4 is limiting metastatic development.

[0009] Unexpectedly, the Inventors discovered that Ackr4 is a target gene of p53, a major tumor suppressor protein, and that the p53-mediated transactivation is inhibited by estrogens. Then, the Inventors demonstrated that Ackr4 is a male-specific biomarker of good prognosis for human Burkitt lymphomas. In a main aspect, the invention thus relates to the in vitro or ex vivo use of a level of expression of Ackr4 as a biomarker for prognosing clinical outcome in a male subject suffering from a B-Cell NonHodgkin lymphoma.

[0010] In the present invention, the term "Ackr4" (or ACKR4) designates a gene encoding a receptor for chemokines CCL2, CCL8, CCL13, CCL19, CCL21 and CCL25. It is also known as "PPR1", "CCBP2", "CCR10", "CCR11" , "CCRL1", “VSHK1", “CCR-11", “CKR-11", "CCX CKR", "CCX-CKR" or “CC-CKR-11".

[0011] For example, some reference sequences of the human Ackr4 gene, its mRNA and protein thereof are provided in Table 1.

[0012] Table 1. Reference sequences of the Ackr4 gene (CDS), mRNA and protein thereof.

[0013] As used herein, the term "biomarker" is intended to mean a measurable indicator of the level of a gene or a gene product thereof, and includes mRNA level, gene copy number, protein level, or the level of a peptide which is associated with a protein of interest. It is a feature that is measured objectively and evaluated as a molecular indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.

[0014] It may be determined as a single value or a range of values which is determined based on the expression level of Ackr4 measured, for instance, in a healthy subject or a subject suffering from a cancer. Preferably, the term "biomarker" refers to the Ackr4 mRNA level. Indeed, the detection of ACKR4 mRNA from bodily fluids presents the advantage to be both particularly sensitive and specific. Alternatively, ACKR4 protein can be detected by using commercial antibodies, e.g. by ELISA. However, this kind of assay exhibits sensitivity limits and risks of cross-reactivity with other proteins which are limiting factors.

[0015] According to the present invention, a high or positive level of expression of Ackr4 in comparison with a reference value is a biomarker allowing the prediction of a positive (or good) clinical outcome, whereas a low or negative level of expression of Ackr4 in comparison with a reference value is a biomarker allowing the prediction of a negative (or poor) outcome.

[0016] As used herein, the expressions "positive outcome" or "good outcome" refer to an increased likelihood of survival within a predetermined period and / or a decreased likelihood of metastases and / or a decreased likelihood of aggravation of the lymphoma after primary treatment.

[0017] As used herein, the expressions "negative outcome" or "poor outcome" refer to a decreased likelihood of survival within a predetermined period and / or an increased likelihood of metastases and / or an increased likelihood of aggravation of the lymphoma after primary treatment.

[0018] As used herein, the term "subject" (or "patient") refers to an animal, preferably to a mammal, even more preferably to a human, including adult, child and elderly person.

[0019] Where a feature is disclosed with respect to an aspect of the invention (e.g. the use of a biomarker), such disclosure is also to be considered to apply mutatis mutandis to any other aspect of the invention (e.g. a method for predicting clinical outcome).

[0020] In another aspect, the invention relates to a method for predicting clinical outcome in a male subject suffering from a B-Cell Non-Hodgkin lymphoma, comprising: i) determining in a biological sample obtained from the subject a level of expression of Ackr4, ii) comparing the level of expression determined at step i) with a reference value, wherein an increase of the level of expression of Ackr4 compared to the reference value is indicative of a positive clinical outcome, said positive clinical outcome being selected from the group consisting of an increased likelihood of survival within a predetermined period, a decreased likelihood of metastases and a decreased likelihood of aggravation of the lymphoma after primary treatment.

[0021] As used herein, the expression "biological sample" designates any sample containing cells derived from a subject, preferably a sample which contains nucleic acids. In particular, the expression "biological sample" may also refer to any sample containing free circulating nucleic acids. The sample may be treated prior to its use, e.g. extracted, purified, concentrated, frozen, etc.

[0022] In an embodiment, the biological sample is selected from the group consisting of blood, serum, plasma, lymph, biopsies, organs, tissues, cell samples and cancer ascites fluids, preferably blood. It may also refer to any sample comprising cancerous cells derived from a patient.

[0023] The level of expression crf Ackr4 can be measured by any method allowing the quantification of the levels of protein or mRNA produced by this gene. Such methods are well known by the skilled person in the art, e.g. Western blotting, ELISA or RT-qPCR.

[0024] As used herein, the expression "reference value" refers to a predetermined value. As the skilled artisan will appreciate, the reference level is predetermined and set to meet the requirements in terms of e.g. specificity and / or sensitivity. These requirements can vary. It may for example be that assay sensitivity or specificity, respectively, has to be set to certain limits. These requirements may also be defined in terms of positive or negative predictive values. In one embodiment, the reference level is determined in healthy individuals. In another embodiment, the reference value is determined in the disease entity to which the patient belongs. In certain embodiments, the reference level can e.g. be set to any percentage between 25% and 75% of the overall distribution of the values in a disease entity investigated. In other embodiments the reference level can e.g. be set to the median, tertiles or quartiles as determined from the overall distribution of the values in a disease entity investigated. In one embodiment, the reference level is set to the median value as determined from the overall distribution of the values in a disease entity investigated.

[0025] In an embodiment, the invention relates to the method as defined above for predicting clinical outcome in a male subject suffering from a B-Cell Non-Hodgkin lymphoma, comprising: i) determining in a biological sample obtained from the subject a level of expression of Ackr4, ii) comparing the level of expression determined at step i) with a reference value, wherein:

[0026] - an increase of the level of expression of Ackr4 compared to the reference value is indicative of a positive clinical outcome, said positive clinical outcome being selected from the group consisting of an increased likelihood of survival within a predetermined period, a decreased likelihood of metastases and a decreased likelihood of aggravation of the lymphoma after primary treatment, and

[0027] -a decrease of the level of expression of Ackr4 compared to the reference value is indicative of a negative clinical outcome, said negative clinical outcome being selected from the group consisting of a decreased likelihood of survival within a predetermined period, an increased likelihood of metastases and an increased likelihood of aggravation of the lymphoma after primary treatment.

[0028] In an embodiment, the invention concerns a method as defined above, wherein the B-Cell NonHodgkin lymphoma is a MYC-driven B-cell lymphoma, preferably a B-cell lymphoma associated to a MYC translocation.

[0029] Indeed, MYC is a potent oncogene initially identified as the target of the t(8;14)(q24;q32) chromosome translocation in Burkitt lymphoma. MYC gene alterations have been identified in other mature B-cell neoplasms that are usually associated with an aggressive clinical behavior. The oncogenic events leading to MYC up-regulation seem to overcome the inhibitory effect of physiological repressors such as BCL6 or BLIMP1. Aggressive lymphomas frequently carry additional oncogenic alterations that cooperate with MYC dysregulation, likely counteracting its proapoptotic function. MYC gene alterations in large B-cell lymphomas are frequently associated with BCL2 or BCL6 translocations conferring a very aggressive behavior. Conversely, MYC protein up-regulation may occur in tumors without apparent gene alterations, and its association with BCL2 overexpression also confers a poor prognosis.

[0030] In an embodiment, the B-Cell Non-Hodgkin Lymphoma is selected from the group comprising Burkitt lymphoma and diffuse large B-cell lymphoma.

[0031] In the most preferred embodiment, the B-Cell Non-Hodgkin Lymphoma is Burkitt lymphoma.

[0032] In an embodiment, the level of expression ofAckr4 is determined by measuring the gene expression of Ackr4. Optionally, the gene expression is measured using a microarray analysis, a quantitative reverse transcription polymerase chain reaction (RT-qPCR), a Northern blot, a serial analysis of gene expression (SAGE) or Next-Generation Sequencing (NGS), preferably by NGS.

[0033] In an embodiment, the level of expression of Ackr4 is determined by using an antibody that specifically binds the ACKR4 protein. Optionally, the ACKR4 protein is detected by an immunoassay selected from a competitive binding assay, a non-competitive binding assay, a radioimmunoassay, an enzyme linked immunosorbent assay (ELISA), a sandwich assay, a precipitin reaction, a gel diffusion immunodiffusion assay, an agglutination assay, a fluorescent immunoassay or a chemiluminescence immunoassay.

[0034] In an embodiment, the increase of the level of expression of Ackr4 in the biological sample is an increase of at least 10%, preferably at least 15% %, more preferably at least 50%. In an embodiment, the increase of the level of expression of Ackr4 in the biological sample is an increase of at least 10 %, 20% 30% 40°% 50% 60% 70% 80% 90% 100% 150% 200% 250% 300% 350% 400% 450% 500% 550% or 600%.

[0035] In some embodiments, the increase of the level of expression of Ackr4 in the biological sample is an increase between 15% and 600%.

[0036] In another aspect, the invention relates to a method for monitoring the effect of a therapy in a male subject suffering from a B-Cell Non-Hodgkin lymphoma and being treated with said therapy, comprising: i) determining in a biological sample obtained from the subject a level of expression ofAckr4, and ii) comparing the level of expression determined at step i) with a reference value corresponding to the level of expression crfAckr4 determined in a sample from said subject at an earlier point of time, wherein an increase of the level of expression crfAckr4 compared to the reference value is indicative that the subject is responsive to said therapy and a decrease of the level of expression of Ackr4 compared to the reference value is indicative that the subject is not responsive to said therapy.

[0037] In another aspect, the invention relates to a method for monitoring the progression of a B-Cell NonHodgkin lymphoma in a male subject, comprising: i) determining in a biological sample obtained from the subject a level of expression ofAckr4, and ii) comparing the level of expression determined at step i) with a reference value corresponding to the level of expression crfAckr4 determined in a sample from said subject at an earlier point of time, wherein a decrease of the level of expression of Ackr4 compared to the reference value is indicative that the lymphoma is progressing and an increase of the level of expression of Ackr4 compared to the reference value is indicative that the lymphoma is not progressing or is shrinking.

[0038] In another aspect, the invention relates to a method for prognosing and treating a male subject suffering from a B-Cell Non-Hodgkin lymphoma, comprising: i) determining in a biological sample obtained from the subject a level of expression ofAckr4, ii) comparing the level of expression determined at step i) with a reference value, wherein:

[0039] - an increase of the level of expression ofAckr4 compared to the reference value is indicative of a positive clinical outcome, said positive clinical outcome being selected from the group consisting of an increased likelihood of survival within a predetermined period, a decreased likelihood of metastases and a decreased likelihood of aggravation of the lymphoma after primary treatment, and

[0040] - a decrease of the level of expression crfAckr4 compared to the reference value is indicative of a negative clinical outcome, said negative clinical outcome being selected from the group consisting of a decreased likelihood of survival within a predetermined period, an increased likelihood of metastases and an increased likelihood of aggravation of the lymphoma after primary treatment,

[0041] (iii) administering an appropriate anti-cancer treatment to the subject .

[0042] Advantageously, in the case of a positive clinical outcome, the appropriate anti-cancer treatment is not an aggressive chemotherapy. At the opposite, in the case of a negative clinical outcome, an aggressive chemotherapy can be required.

[0043] In another aspect, the invention relates to the ACKR4 protein for use as a medicament.

[0044] Indeed, the Inventors unexpectedly demonstrated that the ACKR4 protein hinders MYC-driven B- cell lymphomagenesis.

[0045] In another aspect, the invention thus relates to the ACKR4 protein for use in the treatment or the prevention of a B-Cell Non-Hodgkin lymphoma.

[0046] The invention also relates to a method of treating or preventing a B-Cell Non-Hodgkin lymphoma comprising administering an effective amount of ACKR4 protein to a patient in need thereof.

[0047] The invention also relates to the use of ACKR4 protein in the manufacture of a medicament for the treatment or the prevention of a B-Cell Non-Hodgkin lymphoma.

[0048] In an embodiment, the invention relates to the ACKR4 protein for its use as defined above to inhibit or delay MYC-driven B-Cell Non-Hodgkin lymphomagenesis.

[0049] In an embodiment, the ACKR4 protein inhibits CCL21-mediated cell migration.

[0050] In a preferred embodiment, the B-Cell Non-Hodgkin Lymphoma is selected from the group comprising Burkitt lymphoma and diffuse large B-cell lymphoma.

[0051] In the most preferred embodiment, the B-Cell Non-Hodgkin Lymphoma is Burkitt lymphoma.

[0052] The following Examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the Inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

[0053] FIGURES

[0054] Figure 1. Male-specific acceleration of Myc-induced B-cell lymphomagenesis in mice lacking p53- AS isoforms.

[0055] (A) Tumor-free survival of p53+ / +Ep-Myc and p53AAS / AASEp-Myc mice, classified according to sex. (n=cohort size). (B) Tumor volumes upon dissection of p53+ / +Ep-Myc and p53AAS / AASEp-Myc mice, classified according to sex. Means ± SEM from 100 lymph nodes from p53+ / +Ep-Myc males, 96 from p53+ / +Ep-Myc females, 148 from p53AAS / AASEp-Myc males and 124 from p53AAS / AASEp-Myc females. (C) Myc mRNA and protein levels in lymph node tumors. (D) Levels of p53-a and p53-AS transcripts and p53 protein levels in lymph node tumors. (E) Transcript levels of the indicated p53 target genes. Means ± SEM (n=6 per genotype). (F-G) Apoptosis (F) and cell proliferation (G) in tumor lymph nodes from Ep-Myc males were determined by immunohistochemistry with antibodies against cleaved caspase-3 and ki67, respectively. Positive cells were counted and normalized to the analyzed areas. Means ± SEM (n=6 mice per assay and genotype). In F, G scale bars=50 pm. Statistical analyses with Mantel-Cox (A) and Student's t (B-G) tests. ***P<0.001, *P<0.05, ns: nonsignificant.

[0056] Figure 2. The loss of p53-AS isoforms affects Ackr4 expression in Ep-Myc male mice.

[0057] (A) B-cell subpopulations in spleens of 6 weeks-old p53+ / +Ep-Myc and p53AAS / AASEp-Myc mice. Means ± SEM (n=6 per genotype). (B-C) RNAseq analysis of spleens from p53+ / +Ep-Myc (n=3) and p53AAS / AASEp-Myc (n=4) 4-6 weeks-old male mice. Volcano plot (B), with differentially expressed genes (DEGs) in light grey. Unsupervised clustering heat-map plot (C), with DEGs ranked according to mean fold changes, and protein-coding genes in bold. (D) RTqPCR analysis of candidate DEGs from spleens of p53+ / +Ep-Myc males and p53AAS / AASE .-l\ / lyCmales. Means ± SEM (n=3-4 per genotype). (E) RT-qPCR analysis of indicated DEGs from spleens of 4-6 weeks-old p53+ / + Ep-Myc males, p53AAS / AASE .-MyCmales, p53+ / +Ep-Myc females and p53AAS / AASEpi-Myc females. Means ± SEM (n=3-4 per sex and genotype). (F) Ackr4 is transactivated by p53 in response to stress. Ackr4 mRNAs in untreated or doxorubicin-treated WT and p53 / _MEFs. Data from 2-3 MEFs per genotype. (G) A putative p53 response element in Ackr4 intron 1. Top: Map of the Ackr4 gene, (boxes: exons (grey box: translated region), black line: intron 1); middle: p53 ChIP in doxorubicin-treated MEFs according to ChlP-Atlas (SRX270554); bottom: p53 Response Element (p53RE) consensus sequence (R=G or A, W=A or T, Y=C or T), the putative p53RE and its mutated counterpart. (H) Luciferase assays of the candidate p53RE. A 1.5 kb fragment containing the WT or mutant p53RE was cloned upstream a luciferase reporter, then transfected into p53 / _MEFs together with an expression plasmid for full length p53 (FL), p53-AS or the DNA-binding mutant p53R270H(RH). (I) In MEFs, p53 activation leads to an increased Ackr4 expression attenuated by estradiol. Ackr4 and Cdknla mRNAs were quantified by RT-qPCR from p53+ / +and p53AAS / AASMEFs, untreated or treated with 10 pM Nutlin and / or 5 pg / ml 17-b estradiol (E2). Means ± SEM from 4 independent experiments. (J) Evidence for Myc binding at the Mt2 promoter in B-cells. ChlP-Atlas reports Myc binding to Mt2 promoter sequences in primary B-cells from the lymph nodes of Ep-Myc mice (SRX353785, SRX353783) and in Ep-Myc-induced lymphoma cells (SRX522383). Chr: chromosome. (K) Gene set enrichment analysis (GSEA). GSEA, performed in p53+ / +Ep-Myc and p53AAS / AASEp-Myc male splenic cells, indicated an enrichment of hallmark Myc targets in p53AAS / AASEp-Myc cells. In A, D, E, F, H, I ***P<0.001, **P<0.01, *P<0.05,0P< 0.057, ns: non-significant in Students t or Mann-Whitney tests.

[0058] Figure 3. ACKR4 is a male-specific prognostic factor in Burkitt lymphoma.

[0059] (A) In human cells, p53 activation leads to an increased ACKR4 expression abrogated by estradiol. ACKR4 and CDKN1A mRNAs were quantified by RT-qPCR from p53-proficient (MRC5) and p53- deficient (MRC5-SV40) human fibroblasts, untreated or treated with Nutlin and / or estradiol (E2). Means ± SEM from 4 independent experiments. (B-C) Analysis of lymphoma dataset #GSE4475. ACKR4 gene expression was plotted for all lymphoma patients with clinical follow-up (91 men, 68 women), classified according to sex (B, left). Gene expression (B, right) or survival curves (C) were plotted for the 30% patients (27 men, 20 women) with the highest or lowest ACKR4 expression, classified according to sex. (D-E) Analysis of Burkitt lymphoma-specific dataset #phs00235. ACKR4 gene expression was plotted for all patients with a Burkitt lymphoma diagnosed at age 0-17 (48 males, 29 females), classified according to sex (D, left). Gene expression (D, right) or survival curves (E) were plotted for the 30% patients (15 men, 9 women) with the highest or lowest ACKR4 expression, classified according to sex. (F) The knockout of ACKR4 in Burkitt lymphoma Raji cells increases their CCL21-guided migration. Chemotaxis was assayed by using Boyden chambers with bare polycarbonate membranes. Equal number of cells were deposited on the membrane of a transwell insert, then migration was determined by counting cells in the lower compartment, after 15h of culture with or without CCL21 added to the lower chamber. Statistical analyses by Student's t or Mann-Whitney tests (A, B, D, F) and Mantel-Cox (C, E) test. ***P<0.001, **P<0.01, * P<0.01, °P=0.054, ns: nonsignificant.

[0060] Figure 4. Further analyses of human B-cell lymphoma or Multiple myeloma datasets.

[0061] (A) MT2A gene expression is not a prognostic marker in human B-cell lymphomas. Survival curves for the 30% patients with the highest MT2A mRNA levels and the 30% patients with the lowest MT2A mRNA levels according to sex (n=cohort sizes), for patients from dataset #GSE4475. (B) ACKR4 is a prognostic factor in Burkitt lymphomas but not in diffuse large B-cell lymphomas. Survival curves of patients from dataset #GSE181063, for the 30% patients with the highest ACKR4 mRNA levels and the 30% patients with the lowest ACKR4 mRNA levels, classified according to sex (n=cohort sizes), and diagnosed with either a diffuse large B-cell (top) or a Burkitt (bottom) lymphoma. (C) ACKR4 is not a prognostic factor in Multiple Myeloma. Survival curves of patients from dataset #GSE136337, for the 30% patients with the highest ACKR4 mRNA levels and the 30% patients with the lowest ACKR4 mRNA levels in malignant plasma cells, classified according to sex (n=cohort sizes). Statistical analyses in all panels by Mantel-Cox tests.

[0062] Figure 5. CCR7 is not a reliable prognostic factor in Burkitt lymphoma or DLBCL.

[0063] (A) Analysis of lymphoma dataset #GSE4475. Survival curves were plotted for the 30% patients (27 men, 20 women) with the highest or lowest CCR7 expression, classified according to sex. (B) Analysis of Burkitt lymphoma-specific dataset #phs00235. Survival curves were plotted for the 30% patients (15 men, 9 women) with the highest or lowest CCR7 expression, classified according to sex. (C) Analysis of dataset #GSE181063, comprising patients with Burkitt lymphomas or diffuse large B-cell lymphomas (DLBCL). Survival curves of patients from dataset #GSE181063, for the 30% patients with the highest CCR7 mRNA levels and the 30% patients with the lowest CCR7 mRNA levels, classified according to sex, and diagnosed with either a Burkitt lymphoma (40 men, 10 women) (left) or with a DLBCL (368 men, 322 women) (right). The combined analysis of these 3 datasets does not identify CCR7 as a reliable prognostic factor, neither in men nor in women.

[0064] EXAMPLES mice

[0065] TP53, the human gene for tumor suppressor p53, encodes several isoforms owing to distinct promoters, alternative splicing and multiple translation initiation sites. p53 alternative isoforms can be abnormally expressed in cancer cells and some may regulate the canonical p53 protein. Like its human TP53 homolog, the murine Trp53 gene encodes multiple isoforms differing in their N- or C- termini. p53 isoforms with distinct C-termini result from the splicing of two mutually exclusive final exons: exon 11, encoding the canonical "a" C-terminal domain, and the Alternatively Spliced (AS) exon, encoding another C-terminus. In adult mice, isoforms with the canonical C-terminus are predominant in all tissues. Previously, it was shown that two models (p53A31and p53ACTD), designed to study the consequences of a loss of the canonical p53 C-terminus, exhibited signs of increased p53 activity leading to a rapidly lethal anemia. To determine the role of p53-AS isoforms in vivo, p53AAS, a mouse model with a specific deletion of the AS exon, was created, it was shown that in mouse embryonic fibroblasts (MEFs), the Trp53MSallele prevented the expression of isoforms with the AS C-terminus whereas it did not affect RNA levels for p53 isoforms with the canonical C- terminus. This model was also used to show that p53-AS isoforms had no role in the anemia affecting p53A31 / A31mice.

[0066] In the present invention, the role of p53-AS isoforms in lymphomagenesis was investigated. Spontaneous tumor onset was compared between WT and p53AAS / AASlittermates for over 2 years and no significant difference was observed in tumor-free survival. Because lymphoma is a common neoplasm in C57BL6 / J WT mice and the mouse cohorts used in the present analyses resulted from >10 generations of backcrosses with C57BL6 / J mice, evidences of lymphoma were searched in the lymph nodes and spleen, by macroscopic examination at autopsy and histological analyses. B-cell lymphomas were observed in about 30% of mice of either genotype. In p53+ / +mice, a higher incidence of B-cell lymphomas was observed in females. By contrast, no obvious sex-specific bias was observed for B-cell lymphomas in p53AAS / AASmice, raising the possibility that the loss of p53-AS isoforms affected B-cell lymphomagenesis. However, the numbers of lymphoma-bearing mice were too small to be conclusive.

[0067] Then, Ep-Myc transgenic mice, which are prone to highly penetrant B-cell lymphomas, were used. p53+ / +Ep-Myc and p53AAS / AASEp-Myc mice developed B-cell lymphomas with similar survival curves when sexes were not considered. Importantly however, death was accelerated, and tumor lymph nodes were larger, in p53AAS / AASEp-Myc males compared to their p53+ / +Ep-Myc male counterparts, whereas no difference in lymphomagenesis was noticeable between p53aAS / aASEp-Myc and p53+ / +Ep-Myc female mice (Figure 1A-B). These data (Figure 1A-B), together with the fact that B-cell lymphomas occur with a higher incidence in WT C57BI6 / J female mice 25, suggested that p53+ / +Ep- Myc male mice are more refractory to B-cell lymphomas, and that p53-AS isoforms might confer this male-specific protection against lymphomagenesis.

[0068] 2 - Cause for accelerated lymphomagenesis in p53aAS / aASEp-Myc males

[0069] The mechanisms underlying the accelerated lymphomagenesis in p53AAS / AAS Ep-Myc males were investigated. Inactivating p53 mutations were not more frequent in tumors from p53aAS / aASEp-Myc males than in those from p53+ / +Ep-Myc males, ruling out additional mutations at the Trp53 locus as potential causes for accelerated lymphomagenesis in p53aAS / aASEp-Myc males. Then, a subset of tumors with no detectable Trp53 mutation was analyzed in males of both genotypes and it was found, at RNA and protein levels, that Myc was expressed at similar levels in all tumors (Figure 1C). No difference in p53-a mRNA levels was observed in tumors from both genotypes, although a decrease at the protein level was detected in most tumors from p53aAS / aASEp-Myc males (Figure ID). Nevertheless, similar transcript levels for classical p53 target genes were observed in tumor cells of both genotypes (Figure IE). To test whether a higher tumor volume in p53aAS / aASEp-myc males might result from lower apoptosis and / or higher cell proliferation, tumors were analyzed by immunohistochemistry with antibodies against cleaved caspase-3 or ki-67, respectively. Similar apoptotic and proliferation indexes were observed for both genotypes (Figure 1F-G). In sum, classical assays for p53 activity in tumors failed to account for differences between the two male genotypes.

[0070] The speed of lymphomagenesis in Ep-Myc mice correlates with the extent of B-cell expansion in the first stages of B-cell differentiation and p53 was previously proposed to control the pool of early B cells. Therefore, the levels of the early pre-B / immature B-cells was determined in 6 weeks-old mice, before any sign of tumor. The spleen, a preferential site of B-cell expansion with a relatively high AS / a isoform ratio, was analyzed. Flow cytometry with a combination of markers was used to discriminate the pre-B, immature, transitional and mature B subpopulations. As expected, high numbers of pre-B and immature B-cells were observed in Ep-Myc mice. In males, pre-B and immature B-cells were more abundant in p53aAS / aASEp-Myc animals, while no difference was observed for transitional and mature B-cells (Figure 2A). By contrast, in the spleen of p53+ / +and p53aAS / aAS6 weeks-old male mice without the Ep-Myc transgene, most B-cells were mature B-cells. In Ep-Myc females, the numbers of pre-B and immature B-cells were similar between genotypes, as were the numbers of transitional and mature B-cells (Figure 2A). Interestingly, p53+ / +Ep-Myc males, which develop lymphomas less rapidly, exhibited the lowest number of immature B-cells (Figure 2A), suggesting a direct correlation between the level of immature B-cell expansion and the speed of lymphomagenesis. Together, these data suggested that p53-AS isoforms may not be required to control the pool of early B-cells under normal conditions, but that in an Ep-Myc context they would limit the expansion of pre-tumor early B cells, specifically in males.

[0071] Example 3 - Transcriptomes from p53+ / +Ep-Myc and p53AAS / AASEp-Myc male spleens

[0072] Bulk RNA-seq and differential expression analyses comparing the spleens from 4-6 weeks-old p53AAS / AASEp-Myc males to spleens from age-matched p53+ / +Ep-Myc males were performed. This revealed a limited number of significantly deregulated genes (Figure 2B), including 13 proteincoding genes and 11 pseudogenes (Figure 3C). Out of the 13 protein-coding genes, 10 genes not encoding an immunoglobulin were investigated and the same samples were analyzed by RT-qPCR (Figure 2D). For 6 of the 10 genes, expression levels were too low to be quantified (TcstvS), or differences in expression were not statistically significant (Masp2, Akrlcl9, Cd300lh, H5ra, Slc26al). Of note, Il5ra belonged to this group, although it is regulated by p53, which illustrates the difficulty to analyze subtle effects in our experiments. Taking this into account, the 4 remaining genes, were considered as potentially interesting because they exhibited differences in mRNA levels with statistical significance (p<0.05) or borderline statistical significance (p=0.057): Ackr4, less expressed in p53AAS / AASEp-Myc males, and Faml32a, Mt2 and Prss50, with an increased expression in p53AAS / AAS Ep-Myc males (Figure 2D). Importantly, survival curves indicated that the Myc-induced lethality was delayed in p53+ / +Ep-Myc males compared to p53aAS / aASEp-Myc males, p53+ / +Ep-Myc females and p53AAS / AASEp-Myc females. Thus, transcripts for Ackr4, Faml32a, Mt2 and Prss50 were quantified in the spleen of 4-6 weeks-old p53+ / +Ep-Myc and p53MS / AASEp-Myc females, then compared mRNA levels in p53+ / +Ep-Myc males versus the 3 other groups. Significantly higher expression of Ackr4 and lower expression of Mt2 were found in p53+ / +Ep-Myc males (Figure 2E).

[0073] Ackr4 encodes the atypical chemokine receptor 4, a decoy receptor promoting the degradation of chemokines that modulate cancer cell proliferation and metastasis. The above data suggested that Ackr4 might be a gene transactivated by p53-a and / or p53-AS isoforms. Consistent with this, by extracting data from a transcriptome-wide study in MEFs 33, evidence was found for a p53- dependent transactivation of Ackr4 in response to doxorubicin (Figure 2F). Furthermore, ChlP- Atlas, the database of chromatin immunoprecipitation experiments, indicated p53 binding to sequences within the intron 1 of Ackr4 in doxorubicin-treated MEFs, and a candidate p53 responsive element in this intron was identified (Figure 2G). Luciferase assays were used to show that this p53 responsive element can be bound and regulated by both p53-a and p53-AS (Figure 2G-H). Together, these data show that Ackr4 is indeed a p53 target gene, although RNAseq data indicated that it is expressed at much lower levels than classical p53 targets like Cdknla or Mdm2 in the splenocytes of Ep-Myc mice. Furthermore, Ackr4 was previously shown to be regulated by Foxl2 and estrogen signaling in ovarian cells and 17-b estradiol was recently found to regulate ACKR4 expression in meniscal cells from both sexes, albeit differentially. Accordingly, it was observed, in both WT and p53AAS / AASMEFs, that p53 activation with the Mdm2 antagonist Nutlin led to the transactivation of Ackr4, but that a concomitant treatment with 17-p estradiol markedly decreased, or completely abrogated, Ackr4 transactivation (Figure 21). By contrast, Cdknla was efficiently transactivated under both conditions in mutant and WT cells (Figure 21). These data indicate that Ackr4 is a p53 target gene whose p53-mediated transactivation can be inhibited by estrogens.

[0074] The Mt2 gene, encoding the potentially oncogenic metallothionein-2, was less expressed in p53+ / +Ep-Myc male pre-tumoral splenocytes, which raised the possibility of its direct or indirect repression by p53, potentially through the binding of p53 or the DREAM complex at its promoter. However, ChlP-Atlas reported no binding of these proteins at the Mt2 promoter. Alternatively, evidence that Myc may impact on Mt2 expression was previously obtained, and ChlP-Atlas reported Myc binding at the Mt2 promoter in primary B-cells from lymph nodes of Ep-Myc mice as well as Ep-Myc-induced lymphoma cells (Figure 2J). This suggest that the differential expression of Mt2 in the p53+ / +pre-tumoral splenic cells of Ep-Myc males might result from subtle differences in Myc signaling. Consistent with this, the comparison of transcriptomes of splenic cells from p53AAS / AASEp- Myc males and p53+ / +Ep-Myc males, when analyzed by gene set enrichment analysis, revealed an enrichment of hallmark Myc target genes in p53AAS / AASEp-Myc male splenic cells (Figure 2K).

[0075] 4 - Relevance of ACKR4 expression in Burkitt lymphomas

[0076] Murine and human alternatively spliced p53 isoforms exhibit structural differences and the ChlP- Atlas database does not report p53 binding to the human ACKR4 intron 1. Nevertheless, we found that p53 activation in human cells also led to an increased ACKR4 expression abrogated by 17-p estradiol, whereas 17- estradiol had no significant effect on the p53-mediated transactivation of CDKN1A (Figure 3A). The potential relevance crf ACKR4 expression in human B-cell lymphomas was thus investigated. Public databases of B-cell lymphoma patients with clinical and gene expression information were analyzed. #GSE4475, a dataset of mature aggressive B-cell lymphomas previously used to define Burkitt lymphoma-like specific transcriptomes, comprising 159 patients (91 men, 68 women) with clinical follow-up, was first analyzed. Overall, ACKR4 gene expression was not significantly different between male and female patients (Figure 3B, left). However, average mRNA levels appeared higher in males when the 30% patients of each sex with the highest ACKR4 expression were considered (Figure 3B, right). Strikingly, when the survival of 30% patients with the highest ACKR4 mRNA levels was compared to the survival of the 30% patients with the lowest ACKR4 mRNA levels, high ACKR4 expression correlated with a better prognosis in men, but not in women (Figure 3C). By contrast, for MT2A, the human homolog of Mt2, differences in mRNA levels did not correlate with significant differences in survival for either sex (Figure 4A).

[0077] #GSE181063 dataset was analyzed, it comprises mostly diffuse large B-cell lymphomas (DLBCL; 613 men, 536 women) and a few Burkitt lymphomas (65 men, 18 women). No difference was found in survival curves of DLBCL patients with low versus high ACKR4 levels, neither in men nor in women. However, there was again an increased survival for Burkitt lymphoma male patients with high ACKR4 expression, but not for women (Figure 4B).

[0078] #phs000235, a Burkitt lymphoma-specific dataset (65 men, 37 women) comprising mostly patients diagnosed at 0-17 years of age, was also analyzed. It provides cohorts homogeneous for both tumor type and age of onset. Again, ACKR4 was expressed at higher levels in a subset of male patients (Figure 3D) and high ACKR4 expression correlated with a better prognosis only in males (Figure 3E).

[0079] Finally, #GSE136337, a dataset comprising data from the malignant plasma cells of patients with multiple myeloma (260 men, 166 women) was also analyzed and it was found that ACKR4 is not a prognostic factor for this cancer type (Figure 4C). Altogether, these analyses showed that, as in Ep- Myc mice, ACKR4 is a male-specific positive prognostic factor in Burkitt lymphoma, the archetype of MYC-driven B-cell lymphomas.

[0080] Then, it was considered the possibility that ACKR4 might be more than a biomarker, if it acts as a suppressor of MYC-driven B-cell lymphomagenesis. In support of this hypothesis, it is known that ACKR4 scavenges the chemokine CCL21, a ligand of the chemokine receptor CCR7, and that the ACKR4-mediated sequestration of CCL21 may impair the CCR7 signalling cascade, which might lead to decreased MYC activity.

[0081] To test this hypothesis, a CRISPR-Cas9 approach was designed to perform the knockout of ACKR4 in Raji cells. Raji is a Burkitt lymphoma cell line isolated from a 11 years-old male patient. Although p53 is mutated in Raji cells, these cells overexpress MYC and express both ACKR4 and CCR7, suggesting that they might be suitable to evaluate the impact of ACKR4 on Burkitt lymphoma cell behavior, particularly their migratory capacities. Raji cells were transfected with a vector expressing Cas9, a puromycin resistance gene and either of two guide RNAs targeting ACKR4 (or no guide RNA for control), then puromycin-resistant cells were selected and recovered as cellular pools or diluted to isolate cellular clones. Both guide RNAs targeted sequences in ACKR4 mapping upstream an encoded DRY motif essential for signal transduction, so that Cas9-induced DNA breaks would generate knockout alleles. With this strategy, puromycin-resistant clones were obtained from Raji cells, two of which were verified to be ACKR4 KO clones by DNA sequencing. The proliferation and migration capacities of Raji control cells (transfected with the Cas9 expression vector without guide RNAs) and the two independent ACKR4 KO Raji clones identified, cultured for 15 hours in medium supplemented or not with the CCL21 chemokine, were compared. Under these conditions, Raji cells of all genotypes appeared to proliferate similarly. Strikingly however, the KO of ACKR4 led to a 4- fold increase in CCL21-mediated cell migration, consistent with the hypothesis that ACKR4 may hinder MYC-driven B-cell lymphomagenesis (Figure 3F).

[0082] Because ACKR4 may regulate CCR7 signaling, it was decided to test whether or not CCR7 expression might be a prognostic factor in B-cell lymphomas, particularly for Burkitt lymphoma. Thus, the same datasets that allowed for identification of ACKR4 as a male-specific marker of good prognosis were used to test CCR7.

[0083] However, as shown in Figure 5, the analysis of the 3 datasets did not reveal any significant or consistent prognostic value for CCR7 expression in Burkitt lymphoma or DLBCL, neither for men nor for women.

[0084] Materials and methods

[0085] Mice

[0086] Design and construction of the p53AASmouse model was previously described (Simeonova et al., Cell Rep 3, 2046-2058, 2013). A minimum of 10 backcrosses with C57BI6 / J mice of both sexes were performed before establishing the cohorts of p53+ / +and p53AAS / AASlittermate mice used in this study. Mouse genotyping with multiple primer sets confirmed >99% C57BI6 / J genetic background after 10 backcrosses. Cohorts of p53+ / +Ep-Myc and p53AAS / AASEp-Myc mice were established with identical parental origin of the Ep-Myc transgene. For all experiments, mice housing and treatment were conducted according to Institutional Animal Care and Use Committee of the Institut Curie.

[0087] Cells and cell culture Mouse embryonic fibroblasts (MEFs) were isolated from 13.5 days post-coitum embryos and cultured in a 5% CO2 and 3% O2 incubator, in DMEM Glutamax (GIBCO), with 15% FBS (PAN Biotech),

[0088] 100 pM 2-mercaptoethanol (Millipore), 0.1 mM Non-Essential Amino-Acids and Penicillin / Streptomycin (GIBCO) for less than 5 passages, except for 3T3 experiments, performed in a 5% CO2 incubator for 9 passages. Cells were treated for 24h with 0.5 pg / ml Doxorubicin (Sigma- Aldrich), 15 pM Etoposide (Sigma-Aldrich), 10 pM Nutlin 3a (Sigma-Aldrich) and / or 5 pg / ml 17b- Estradiol (Merck). At least 3 independent experiments with at least 2 independent littermate MEF clones of each genotype and each sex were performed to measure DNA damage responses. For estradiol assays, four independent experiments with three independent MEF male clones of each genotype were performed. Human lung fibroblast MRC5 and its SV40-transformed derivatives were cultured in a 5% CO2 and 3% C -regulated incubator in MEM medium without Phenol Red (Gibco), completed with 10% FBS, 2 mM L-glutamine (Gibco), 1 mM pyruvate, 0.1 mM Non-Essential AminoAcids, and penicillin / streptomycin, and treated for 24h with 10 pM Nutlin 3a (Sigma-Aldrich) and / or 5 pg / ml 17b-Estradiol (Merck). Four independent experiments were performed. Burkitt lymphoma cells (Raji or Raji ACKR4 KO derivatives) were cultured in a 5% CO2 incubator in RPMI Glutamax (Gibco) supplemented with 10% FBS, 2 mM L-glutamine, 1 mM pyruvate, 0.1 mM Non-Essential Amino-Acids, 0.45% glucose (Sigma-Aldrich), and penicillin / streptomycin. In proliferation and migration assays, Raji control cells and two independent ACKR4 KO derivatives were treated or not for 15h with 1 mg / ml CCL21 (PreproTech) then counted in triplicates.

[0089] Quantitative RT-PCR

[0090] Total RNAs were extracted using nucleospin RNA II (Macherey-Nagel), reverse-transcribed using superscript IV (Invitrogen), and real-time quantitative PCRs were performed on an ABI PRISM 7500 using Power SYBR Green (Applied Biosystems). For quantification of p53 isoforms in healthy tissues, a forward primer in exon 10 and a reverse primer encompassing the boundary between exons 10 and 11 were used for p53-a amplification, whereas the same forward primer and a reverse primer located in exon AS were used for p53-AS amplification. To determine the AS / a mRNA ratios, expression levels were compared with a standard curve generated by serial dilutions of a plasmid containing both p53-AS and p53-a cDNAs.

[0091] Western blots

[0092] Thymocytes were lysed in RIPA buffer (50 mM Tris-HCI pH 8, 150 mM NaCI, 5 mM EDTA, 0.5% deoxycholic acid, 0.1% SDS, 1% NP-40) with a cocktail of protease inhibitors (Roche) and 1 mM PMSF (Sigma). Whole-cell extracts were sonicated three times for 10 s and centrifuged at 13 000 r.p.m. for 30 min to remove cell debris. MEFs or B-cell lymphomas were lysed in Giordano's buffer (50 mM Tris-HCI pH 7.4, 250 mM NaCI, 5 mM EDTA, 0.1% Triton X-100) with a cocktail of protease inhibitors (Roche) and 1 mM PMSF (Sigma). Protein lysate concentration was determined by BCA assay (Thermo Scientific) and 30 pg of each lysate was fractionated by SDS-PAGE on a 4-12% polyacrylamide gel and transferred onto PDVF membrane (Amersham). Membranes were incubated with antibodies against p53(CM5, Novocastra), myc (9E-10, Santa Cruz), p21 (F-5, Santa Cruz) and actin (actin-HRP sc47778, Santa Cruz) and revealed with SuperSignal West femto detection reagent (Thermo Scientific).

[0093] MEFs with the indicated genotypes were sequentially infected with pWZL-ElA12S and pBABE- Hrasvl2 viruses. In total, 5xl06E1A- and Ras- (ElA+Ras) expressing MEFs of each genotype were injected subcutaneously into the flanks of 7-weeks old female athymic nude mice (at least 4 mice per genotype) and tumor volumes were determined 1, 8, 15, 21 and 25 days after injection. Importantly, populations of (ElA+Ras)-expressing cells were used to minimize potential differences in expression levels that could result from independent viral insertion sites.

[0094] Cell sorting of B-cell sub-populations

[0095] Splenocytes were recovered from 6 weeks-old asymptomatic mice and incubated with DAPI and the following antibodies: APC rat anti-mouse CD45R / B220, FITC rat anti-mouse CD43, PE rat antimouse IgM and BV605 rat anti-mouse IgD (BD Pharmingen). First, theB220+CD43- cells were selected by flow cytometry from DAPI negative living cells, yielding subsequently 4 different B subpopulations based on IgM and IgD labeling: lgM- / lgD- preB lymphocytes, IgM low / IgD- immature B lymphocytes, IgM high / lgD- transitional B lymphocytes and lgM+ / lgD+ mature B lymphocytes.

[0096] RNA-Seq analysis

[0097] Total RNA was extracted from the spleen of 4-6 weeks-old asymptomatic mice using nucleospin RNA II (Macherey-Nagel). The quality of RNA was checked with Bioanalyzer Agilent 2100 and RNAs with a RIN (RNA integrity number) > 6 were retained for further analysis. RNA was depleted from ribosomal RNA, then converted into cDNA librairies using a TruSeq Stranded Total Library preparation kit (Illumina). Paired-end sequencing was performed on an Illumina MiSeq platform. Reads were mapped to the mouse genome version GRCm38 and counted on gene annotation gencode.vM18 with featurecounts. Differentially expressed genes of C57BI6 / J genetic background with an adjusted p-value < 0.05 were identified using the DESeq2 R package. Gene set enrichment analysis was performed by using the GSEA software with canonical pathway gene sets from the Mouse Molecular Signature Database (MSigDB).

[0098] Luciferase assays

[0099] The candidate p53 responsive element (p53 RE) in the Ackr4 promoter was identified by using the JASPAR database of binding profiles with the position weight matrix scanner PWMscan. A 1.5 kb fragment from Ackr4 intron 1, containing a WT or mutant p53 RE at its center, was cloned upstream a SV40 minimal promoter and a luciferase reporter gene in the backbone of a PGL3 plasmid (Promega). Lipofectamine 2000 was used to transfect p53 / _MEFs with 2 pg of either luciferase expression vector, 2 pg of an expression vector for p53WT, p53ASor the DNA-binding mutant p53R270H, and 30 ng of a renilla luciferase expression plasmid (pGL4.73, Promega) for normalization. Transfected cells were incubated for 24 h then trypsinized, resuspended in 75 pl culture medium with 7.5% FBS and transferred into a well of an optical 96-well plate (Nunc). The dual-glo luciferase assay system (Promega) was used according to the manufacturer's protocol to lyse the cells and read firefly and renilla luciferase signals. Results were normalized, then the average luciferase activity in cells transfected with the WT p53RE luciferase reporter and the p53R270Hexpression plasmid were assigned a value of 1.

[0100] Generation crf ACKR4 Knockout Burkitt lymphoma cells

[0101] Six guide RNAs (gRNAs) were designed to target the human ACKR4 gene by using the web tool CRISPOR. For each gRNA, two reverse complementary oligonucleotides were designed (with added backbone sequences, including a 5' G nucleotide for gRNA sequences without a 5' G to improve transcription efficiency from a U6 promoter), annealed and cloned in the Cas9 expression vector pSpCas9(BB)-2A-Puro (PX459) (Addgene). Preliminary tests for efficiency to induce cleavage in the targeted DNA regions were performed in HEK293T cells, which led to select the gRNAs #4 (5'- TGGTAGTGGCAATTTATGCC-3', SEQ ID NO: 8) and #5 (5'-GGGCTGTTAATGCAGTTCAT-3', SEQ ID NO: 9) for further experiments. Raji Burkitt lymphoma cells were transfected with a PX459 vector expressing gRNA #4 or #5 (or no gRNA for control), by using Nucleofector Amaxa kit V (Lonza), and selection was carried out with 1 pg / ml puromycin for 3 weeks to obtain stably transfected cells. Clones were obtained by seeding cells at low density (1 cell / 5 wells) in 96-well plates, expanded, then subdivided in two parts, half for storage in liquid nitrogen and half for ACKR4 genotyping by Sanger DNA sequencing. For each cell clone, the ACKR4 target DNA regions were amplified by PCR, PCR products were cloned in a PGL3 plasmid, and 8 transformed bacteria colonies were sequenced. This led to identify two homozygous ACKR4 KO clones, one (clone 4.14) obtained by using gRNA #4 and one (clone 5.2) by using gRNA #5. Cell mi

[0102] Cell migration assays towards the chemokine CCL21 were performed with Boyden chambers by measuring transwell migration across bare polycarbonate membranes with a pore size of 5 mm (Corning). A total of 100 ml of culture medium (RPMI Glutamax, 10% FBS, 2 mM L-glutamine, 1 mM pyruvate, 0.1 mM Non-Essential Amino-Acids, 0.45% glucose, and penicillin / streptomycin) containing 5xl05cells was added to a 6.5 mm diameter transwell insert, and 600 ml of culture medium with or without lmg / ml CCL21 were added to the lower compartment. After 15h at 37°C in 5% CO2, the number of migrated cells in the lower chamber was determined by using a Coulter Counter.

[0103] Statistical

[0104] Student's unpaired t tests were used in most Figures to analyze differences between WT vs AAS values. Log-Rank Mantel-Cox were used to analyze Kaplan-Meier tumor-free survival curves. Analyses were performed using Graphpad Prism 5, and values of p<0.05 were considered significant.

Claims

CLAIMS1. In vitro or ex vivo use of a level of expression ofAckr4 as a biomarker for prognosing clinical outcome in a male subject suffering from a B-Cell Non-Hodgkin lymphoma.

2. A method for predicting clinical outcome in a male subject suffering from a B-Cell Non-Hodgkin lymphoma, comprising: i) determining in a biological sample obtained from the subject a level of expression of Ackr4, ii) comparing the level of expression determined at step i) with a reference value, wherein an increase of the level of expression o Ackr4 compared to the reference value is indicative of a positive clinical outcome, said positive clinical outcome being selected from the group consisting of an increased likelihood of survival within a predetermined period, a decreased likelihood of metastases and a decreased likelihood of aggravation of the lymphoma after primary treatment.

3. A method for monitoring the effect of a therapy in a male subject suffering from a B-Cell Non-Hodgkin lymphoma and being treated with said therapy, comprising: i) determining in a biological sample obtained from the subject a level of expression of Ackr4, and ii) comparing the level of expression determined at step i) with a reference value corresponding to the level of expression of Ackr4 determined in a sample from said subject at an earlier point of time, wherein an increase of the level of expression of Ackr4 compared to the reference value is indicative that the subject is responsive to said therapy and a decrease of the level of expression of Ackr4 compared to the reference value is indicative that the subject is not responsive to said therapy.

4. A method for monitoring the progression of a B-Cell Non-Hodgkin lymphoma in a male subject, comprising: i) determining in a biological sample obtained from the subject a level of expression of Ackr4, andii) comparing the level of expression determined at step i) with a reference value corresponding to the level of expression of Ackr4 determined in a sample from said subject at an earlier point of time, wherein a decrease of the level of expression of Ackr4 compared to the reference value is indicative that the lymphoma is progressing and an increase of the level of expression of Ackr4 compared to the reference value is indicative that the lymphoma is not progressing or is shrinking.

5. The method according to any one of claims 2 to 4, wherein the B-Cell Non-Hodgkin lymphoma is a M / C-driven B-cell lymphoma, preferably a B-cell lymphoma associated to a MYC translocation.

6. The method according to any one of claims 2 to 5, wherein the B-Cell Non-Hodgkin Lymphoma is selected from the group comprising Burkitt lymphoma and diffuse large B-cell lymphoma, preferably Burkitt lymphoma.

7. The method according to any one of claims 2 to 6, wherein the biological sample is selected from the group consisting of blood, serum, plasma, lymph, biopsies, organs, tissues, cell samples and cancer ascites fluids, preferably blood.

8. The method of any one of claims 2 to 7, wherein the increase of the level of expression ofAckr4 in the biological sample is an increase of at least 10%, preferably 15%, more preferably 50%.

9. The method according to any one of claims 2 to 8, wherein the level of expression of Ackr4 is determined by measuring the gene expression of Ackr4, optionally wherein the gene expression is measured using a microarray analysis, a quantitative reverse transcription polymerase chain reaction (RT-qPCR), a Northern blot, a serial analysis of gene expression (SAGE) or Next-Generation Sequencing (NGS), preferably by NGS.

10. The method according to any one of claims 2 to 9, wherein the level of expression of Ackr4 is determined by using an antibody that specifically binds the ACKR4 protein, optionally wherein the ACKR4 protein is detected by an immunoassay selected from a competitive binding assay, a non-competitive binding assay, a radioimmunoassay, an enzyme linked immunosorbent assay (ELISA), a sandwich assay, a precipitin reaction, a gel diffusion immunodiffusion assay, an agglutination assay, a fluorescent immunoassay or a chemiluminescence immunoassay.

11. ACKR4 protein for use as a medicament.

12. ACKR4 protein for use in the treatment or the prevention of a B-Cell Non-Hodgkin lymphoma.

13. ACKR4 protein for its use according to claim 12, wherein the ACKR4 protein inhibits CCL21- mediated cell migration.

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