WO2019154884A1 - Method for determining cancer invasiveness and patient prognosis - Google Patents

Method for determining cancer invasiveness and patient prognosis Download PDF

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WO2019154884A1
WO2019154884A1 PCT/EP2019/052949 EP2019052949W WO2019154884A1 WO 2019154884 A1 WO2019154884 A1 WO 2019154884A1 EP 2019052949 W EP2019052949 W EP 2019052949W WO 2019154884 A1 WO2019154884 A1 WO 2019154884A1
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gene
cancer
genes
inhibitor
gene product
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French (fr)
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Douglas Hanahan
Leanne LI
Arjun BHUTKAV
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Ecole Polytechnique Federale De Lausanne (Epfl)
Massachusetts Institute Of Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the subject matter described in the present disclosure lies in the field of molecular oncology. More particularly, the subject-matter described in the present disclosure relates to a method for determining and/or improving the prognosis for survival of a cancer patient, said method being based on assessment of the activation profile of the NMDAR signalling pathway that stimulates malignant tumour growth and impacts a cancer patient’s prognosis.
  • Modifier genes can modulate the penetrance of specific driver oncogenes, exerting either protective or detrimental effects and affecting therapeutic outcome.
  • QTL quantitative trait locus
  • GWAS genome-wide association analyses
  • the RIP1 Tag2 transgenic mouse model of pancreatic neuroendocrine tumour recapitulates the multi-stage nature of human cancer progression. As such, it has proved to be a valuable research tool for elucidating mechanisms of tumour development and malignant progression to invasive and metastatic states. Interestingly, varying degrees of tumour invasiveness are observed at end stage depending on the genetic background in which the same transgene integration is resident, despite expressing similar levels of the driving oncoprotein (SV40 T- antigen) under control of the Rat Insulin Promoter (RIP).
  • SV40 T- antigen driving oncoprotein
  • RIP Rat Insulin Promoter
  • the RIP1Tag2 model may phenocopy a facet of the complexity of cancer progression in patients, where the same oncogenetic events can lead to varying outcomes in different patient populations.
  • the identified region harbours more than 50 genes, having no polymorphic differences in their coding regions of these genes. Seven genes were identified as the most differentially expressed in mPanNETs arising in the B6 versus the C3H genetic strain backgrounds, and implicated as potential invasion modifier candidates. The inventors focused later on their attention on dlgap1/GKAP, a key adaptor protein of the glutamate-activated N-methyl-D-aspartate receptors (NMDA receptor, also known as GluNR) in the central nervous system.
  • NMDA receptor glutamate-activated N-methyl-D-aspartate receptors
  • GluNR glutamate-activated N-methyl-D-aspartate receptors
  • GKAP clusters and potentiates NMDAR activity via its interaction with the upstream scaffold protein PSD-95, which directly binds the NMDAR; moreover, it organizes and stabilizes a variety of NMDAR scaffold complexes to transmit signals downstream.
  • NMDAR signalling is active and promotes invasive tumour growth in the pro-invasive B6 background of this mouse model of PanNET, where GKAP is highly expressed (Li and Hanahan, 2013). Nonetheless, how exactly the genetic background of an individual could impact cancer invasiveness and progression, as well as possible gene signatures influencing this complex process, is a controversial argument yet under debate. Methods and compounds to ascertain and modulate cancer invasiveness so to promptly intervene and improve patients’ prognosis are still urgently needed.
  • prognostic markers that can accurately distinguish tumours associated with good prognosis including low probability of metastasis, late disease progression, decreased disease recurrence or increased patient survival, from the others.
  • different signalling pathways may be dominant in different patients, it is only by identifying patients whose tumours have activated specific molecular pathways using such markers, that the practitioner can predict the patient's prognosis and can effectively target the individuals who would most likely benefit from therapy or who need a more intensive monitoring.
  • a method for determining the prognosis for survival of a patient having a cancer said cancer being altered (i.e. highly activated or alternatively inactive/inhibited) in the NMDAR signalling pathway, said method comprising a step of determining the transcription level and/or expression level and/or the activity of a plurality of genes or gene products selected from genes listed in Tables 1 to 4 in a sample from said cancer patient relative to a control, wherein the differential transcription and/or expression and/or activity of said plurality of genes or gene products relative to a control is indicative of cancer aggressiveness and therefore of the patient’s prognosis.
  • Another object of the subject-matter described in this disclosure relates to a method for improving the prognosis for a cancer patient being diagnosed to have a poor prognosis, comprising modulating the transcription level and/or the expression level and/or the activity of:
  • DLGAP1 a gene or gene product member of the DLGAP family of genes (including DLGAP1 , DLGAP2, DLGAP3, DLGAP4 and DLGAP5); and/or
  • said modulating is carried out by administering to said cancer patient:
  • a further object of the subject-matter described in this disclosure relates to a pharmaceutical composition for use in the treatment of a cancer in a patient being diagnosed to have a poor prognosis, comprising: a. (an) inhibitor(s) of at least one gene, or gene product derived therefrom, listed in Tables 2 and/or 4; and/or
  • kits for in vitro analysis aimed at determining the prognosis of a patient having a cancer, said cancer being altered in the NMDAR signalling pathway, said kit comprising a reagent that selectively interacts with one or more of:
  • Figure 1 shows the differential GKAP expression between the C57/BI6 and C3Heb/Fe genetic backgrounds associated with a differential NMDAR pathway activity in vitro:
  • C Among the multiple SNPs associated with the GKAP gene and its flanking sequences that distinguished C3H mice from B6 mice, a transcription factor prediction algorithm identified only one SNP that matched with a potential transcription factor binding site (upper panel). This site in the B6 version of the GKAP gene putatively binds to HSF1 (p ⁇ 0.004).
  • Figure 2 shows that the intracellular calcium responses and electrophysiology reveals functional NMDAR in bTOBQ but not in bT0-03H cells:
  • the top panel shows ⁇ TC-B6 in phase- contrast, whereas the lower panel shows a green-fluorescence signal overlaid with a phase-contrast image,
  • the Y-axis indicates the change in fluorescence intensity;
  • Figure 3 shows that high GKAP expression is associated with increased NMDAR pathway activity in vivo:
  • Figure 4 shows that GKAP regulates cancer cell invasion through NMDAR activity and downstream effectors FMRP and HSF1 :
  • I ⁇ -3 cells Clusters of cultured control « TC-3 cells (i) or GKAP-KD b " I ⁇ -3 cells (ii) were analyzed under a bright field microscope after a NMDA solution (1 mM, 1 s) was puffed through perfusion pipette at left (upper panels). NMDA caused large synchronous calcium transients in a group of Control-KD cells (left panels); in contrast, GKAP-KD cells do not show responses synchronized to the NMDA puff, but rather only small spontaneous, asynchronous transients (right panels).
  • FMRP knockdown (upper panel) decreased bT03 invasion in the flow-guided invasion assay (lower panel).
  • the numbers below indicate levels of FMRP normalized to GAPDH. Unpaired t-test, * : p ⁇ 0.05. ** : p ⁇ 0.01.
  • FIG. 5 shows that NMDAR signaling through GKAP promotes invasion in both mouse and human PDAC cell lines:
  • Figure 6 shows that the NMDAR/GKAP/FMRP/HSF1 signaling axis is active in PDAC tumours:
  • GKAP, FMRP and HSF1 are highly expressed in mPDAC GEMM, both in primary PDAC tumours (upper panels) and in liver metastases (lower panels).
  • the primary tumour panels are representative of >5 tumour fields/pancreas from >20 mice.
  • the liver metastasis panels are representative of two liver macro-metastases ( ⁇ 1cm in diameter);
  • p-GluN2b, GKAP, FMRP and HSF1 were all detected in hPDAC tumours displayed in a tissue microarray (i). The images were quantified to show the percentage of positive areas on each tissue section, and the expression of all four proteins was further elevated in the progression from primary to lymph node metastatic PDAC in humans (ii). The expression of GKAP, FMRP and HSF1 all positively correlated with p-GluN2b expression (iii). p-GluN2b was also associated with larger tumour size (iii) and with vascular invasion by cancer cells (iv), classified as absent (VO) or present (V1);
  • Figure 7 shows the identification of gene expression signatures for mPanNETs:
  • A Schematic presentation for gene expression signatures associated with tumour phenotypes. Bold italic and normal boxes mark the samples used in the signature analysis; bold italic suggests enrichment of the gene expression signature, while normal suggests depletion;
  • Figure 8 shows that the signatures of low-/inhibited- NMDAR activity is associated with favorable prognosis in human cancer types:
  • FIG. 9 shows that GKAP/dlgap1 is the most differentially expressed invasion modifier gene:
  • FIG. 10 shows that FMRP and HSF1 are downstream effectors of the
  • FIG. 12 shows that MK801 has therapeutic benefit in PDAC GEMM; MK801 treatment signature was associated with better survival in patients:
  • TCGA http://cancergenome.nih.gov/.
  • Gene expression data from patient cohorts was stratified by their enrichment for the“NMDAR-pathway l0W signature” identified in the mPanNET RNA-seq analysis.
  • Full line patients whose tumours had gene expression most correlated with the core driver gene signature (defined by the top 3.5 Z score); dotted line: patients whose tumours had gene expression least correlated with the core driver gene signature (defined by the bottom 3.5 Z score). All patients were included in each cancer type shown, regardless of treatment and staging. LLC: low grade glioma.
  • a “gene” is defined as a nucleic acid molecule that comprises a nucleic acid sequence that encodes an RNA molecule and the expression control sequences that surround the nucleic acid sequence that encodes the RNA molecule.
  • the encoded RNA molecule may be a functional RNA (e.g. tRNA, rRNA), regulatory RNA (e.g. siRNA, miRNA, tncRNA, smRNA, snRNA) or messenger RNA (mRNA).
  • Messenger RNA molecules may be transcribed into polypeptides which are also considered as being encoded for by the gene.
  • a gene may comprise a promoter, one or more enhancers, a nucleic acid sequence that encodes an RNA molecule, downstream regulatory sequences and, possibly, other nucleic acid sequences involved in regulation of the expression of an RNA.
  • eukaryotic genes usually contain both exons and introns.
  • exon refers to a nucleic acid sequence found in genomic DNA that is bioinformatically predicted and/or experimentally confirmed to contribute contiguous sequence to a mature RNA transcript.
  • intron refers to a nucleic acid sequence found in genomic DNA that is predicted and/or confirmed to not contribute to a mature RNA transcript, but rather to be "spliced out" during processing of the transcript.
  • a “gene product” is defined as a molecule expressed or encoded directly or indirectly by a gene.
  • gene products include pre-mRNA, mature mRNA, tRNA, rRNA, snRNA, uIRNA, siRNA, miRNA, tncRNA, smRNA, prepolypeptides, pro-polypeptides, mature polypeptides, post translationally modified polypeptides, processed polypeptides, functionally active polypeptides, functionally inactive polypeptides, and complexed polypeptides.
  • a single gene product may have several molecular functions and different gene products may share a single or similar molecular function.
  • the gene product also encompasses variants and/or fragments thereof.
  • the term“variant” refers to biologically active derivatives of a nucleic acid sequence or polypeptide sequence of the invention.
  • the term“variant” refers to molecules having a native sequence and structure with one or more additions, substitutions (generally conservative in nature) and/or deletions (e.g. splice variants), relative to the native molecule, so long as the modifications do not destroy biological activity and which are“substantially homologous” to the reference molecule.
  • the sequences of such variants are functionally, i.e.
  • sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90% or 95% or more, when the two sequences are aligned.
  • the term“variant” also refers to post-transcriptionally modified nucleic acid sequence (e.g. methylation, phosphorylation, etc..) or polypeptide sequence (e.g. isoform, (7) of the invention.
  • a“fragment” of one or more nucleic acid sequence or polypeptide sequence of the invention refers to a sequence containing less nucleotides or amino acids in length than the respective sequences of the invention while retaining the biological activity described herein.
  • this fragment contains, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid sequence or polypeptide sequence.
  • the terms also include genes or gene products whose transcription and/or expression and/or activity is altered to a higher or lower level at different stages of the same disease.
  • differentially transcripted and/or expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alternative splicing to result in a different polypeptide product. Such differences may be evidenced by a change in mRNA levels, surface expression, secretion or other partitioning of a polypeptide, for example.
  • Differential gene transcription and/or expression and/or activity may include a comparison of transcription and/or expression and/or activity between two or more genes or their gene products, or a comparison of the ratios of the transcription and/or expression and/or activity between two or more genes or their gene products, or even a comparison of two differently processed products of the same gene, which differ between normal subjects and subjects suffering from a disease, specifically cancer, or between various stages of the same disease.
  • the reference (“control”) transcription and/or expression and/or activity level can be the transcription and/or expression and/or activity level of the gene product in a control sample.
  • the control sample can be a normal sample, that is, a non-tumoural sample, preferably from the same tissue than the cancer sample, or a basal level of transcription and/or expression and/or activity.
  • the normal sample may be obtained from the subject affected with the cancer or from another subject, such as a normal or healthy subject, i.e. a subject who does not suffer from a cancer.
  • the control sample can be obtained from data repositories of cancer patient expression studies; accordingly, the control sample could be in this case a virtual sample obtained from such a data repository for a given cancer type.
  • Transcription and/or expression and/or activity levels obtained from cancer and normal samples may be normalized by using e.g. expression levels of proteins which are known to have stable expression such as RPLPO (acidic ribosomal phosphoprotein PO), TBP (TATA box binding protein), GAPDH (glyceraldehyde 3- phosphate dehydrogenase) or b-actin. Additionally or alternatively, as it will be appreciated by a person skilled in the art, it is possible to use statistical methods based on whole-transcriptome expression distribution for normalization of cancer and control/normal samples.
  • Differential transcription and/or expression and/or activity includes both quantitative, as well as qualitative, differences in the temporal or cellular transcription and/or expression and/or activity pattern in a gene or its products among, for example, normal and diseased cells, or among cells which have undergone different disease events or disease stages.
  • “differential gene transcription and/or expression and/or activity” is considered to be present when there is at least a two-fold difference between the transcription and/or expression and/or activity of a given gene or gene product in normal and diseased subjects, or in various stages of disease development in a diseased subject.
  • NMDAR-pathway-LOW signatures can be defined in comparison to other tumours of the same type that are NMDAR-pathway-HIGH.
  • RNA transcript is used to refer to the level of the transcript determined by normalization to the level of reference mRNAs, which might be all measured transcripts in the specimen or a particular reference set of mRNAs.
  • percent sequence identity in the context of nucleic acid or amino acid sequences refers to the residues in two sequences which are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over a stretch of at least about nine, usually at least about 20, more usually at least about 24, typically at least about 28, more typically at least about 32, and preferably at least about 36 or more nucleotides or amino acids.
  • a reference to a nucleic acid sequence encompasses its complement unless otherwise specified.
  • a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.
  • percent sequence identity percent sequence similarity
  • percent sequence homology percent sequence homology
  • the terms "patient” or“subject” refer to an animal, preferably to a mammal, even more preferably to a human, including adult and child. However, the terms can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheeps and non-human primates, among others, that are in need of treatment.
  • sample means any sample containing cells, nucleic acids and/or proteins derived from a subject.
  • samples include fluids such as blood, plasma, saliva, and urine as well as biopsies, organs, tissues or cell samples.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Additionally or alternatively, the terms refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Additionally or alternatively, the terms refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • cancer examples include, but are not limited to, blood cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, and brain cancer.
  • a cancer is said to be “aggressive” if it forms, grows, or spreads quickly. Aggressive cancers are characterized by a high malignancy, i.e. marked metastasizing and invasive properties correlated with a poor prognosis. As used herein, a cancer is said to be“invasive” if it has the ability to disrupt and spread beyond a basement membrane, or beyond its confines into adjacent normal tissue. Invasive cancers generally carry a poorer prognosis than non-invasive cancers, since invasive cancers are not delimited by basement membrane barriers and can metastasize to other areas of the body.
  • prognosis defines a forecast as to the probable outcome of a disease, the prospect as to recovery from a disease, or the potential recurrence of a disease as indicated by the nature and symptoms of the case.
  • the term further refers to the prediction of the likelihood of cancer-attributable death or progression, including recurrence, metastatic spread and/or drug resistance, of a neoplastic disease.
  • prediction is used herein to refer to e.g. the likelihood that a patient will respond either favourably or unfavourably to a drug or set of drugs, and also the extent of those responses, or that a patient will survive, following surgical removal or the primary tumour and / or chemotherapy for a certain period of time without cancer recurrence.
  • the predictive described herein can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient.
  • the term "good prognosis” refers to a probable favourable outcome of a disease, recovery from a disease or low potential for disease recurrence such as an increased patient survival and/or a late disease progression and/or a and/or a decreased metastasis formation.
  • treatment generally refers to any act intended to ameliorate the health status of subjects such as an animal, particularly a mammal, more particularly a human, such as therapy, prevention, prophylaxis and retardation of the disease, and includes: (a) inhibiting the disease, i.e., arresting its development; or (b) relieving the disease, i.e., causing regression of the disease and/or its symptoms or conditions such as improvement or remediation of damage.
  • such term refers to the amelioration or eradication of a disease or symptoms associated with a disease.
  • this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.
  • prevention or“preventing” relates to hampering, blocking or avoid a disease from occurring in a subject which may be, for any reason, predisposed to the disease but has not yet been diagnosed as having it for example based on familial history, health status or age.
  • the inventors established a research program aimed at identifying genetic signatures and the influence of the genetic background capable to explain the PanNET mouse model phenotype, as well as their contribution in both animal models and human counterparts in tumour malignancy, namely the capability for tissue invasion (and metastasis).
  • the subject-matter described in the present disclosure is based, at least in part, on the surprising evidence that, in mouse models, GKAP is a potential modifier gene of NMDAR pathway activity whose differential expression contributes to genetic background-specific differences in pancreatic tumour invasiveness.
  • the inventors elucidated the autocrine glutamate-NMDAR-GKAP signalling axis and its functional role in the invasive/malignant phenotypes of both mouse and human pancreatic ductal adenocarcinoma (PDAC).
  • PDAC pancreatic ductal adenocarcinoma
  • the inventors have identified a gene expression signature for inhibited/low NMDAR signalling in the PanNET transcriptome, which is significantly associated with favourable patient survival in several human cancers, including PDAC. This association is useful for predicting the aggressiveness of a cancer and assessing treatment options.
  • the present disclosure therefore provides a set of genes and gene products, the expression, transcription or blocking/inhibition of which is of prognostic value.
  • the subject-matter described in this specification provides for a method for determining the prognosis for survival of a patient having a cancer, said cancer being altered (i.e. highly activated or alternatively inactive/inhibited) for what concerns the NMDAR signalling pathway, said method comprising a step of determining the transcription level and/or expression level and/or the activity of a plurality of genes or gene products selected from genes listed in Tables 1 to 4 in a sample from said cancer patient relative to a control, wherein the differential transcription and/or expression and/or activity of said plurality of genes or gene products relative to a control is indicative of cancer aggressiveness and therefore of the patient’s prognosis.
  • the differential transcription and/or expression and/or activity of said plurality of genes or gene products relative to a control can be up- regulation or down-regulation of said transcription and/or expression and/or activity of said genes or gene products compared to a control level such as for instance a basal level.
  • the "NMDAR signalling pathway”, as referred to herein, refers to a series of biochemical events, as well as to the signalling biochemical elements involved in said events, downstream to the NMDA receptor (“NMDAR”) activation.
  • This signalling pathway is intended to include molecular events from activation of the NMDA receptor to end effects such as e.g. cell proliferation and/or invasion, or intermediate effects such as Ca2+ cell influx or phosphorylation of NMDA receptor subunits.
  • altered in the NMDAR signalling pathway is intended to mean the alteration of one or more signalling elements in the pathway (e.g. to affect its enzymatic, structural or other functional properties) which affects downstream signalling events.
  • Alteration of the signalling elements refers to e.g. the ability to form interactions with other molecules, e.g. protein-protein interactions. The ultimate effect is an alteration of downstream events which typify the NMDAR signalling. Alteration of said signalling pathway may be assessed for example by determining the extent of activation of a molecule involved in said pathway, examination of levels of molecules whose levels are dependent on the activity of said pathway and the like.
  • An altered NMDAR signalling pathway can be a highly activated NMDAR signalling pathway or alternatively an inactive/inhibited or less active NMDAR signalling pathway compared to any suitable control such as a baseline, normal level.
  • a highly activated NMDAR signalling pathway is present in concomitance with a high GKAP expression in tumour cells, which leads to deregulated protein translation and elevated FMRP/p-HSF1 expression, and finally heightens a tumour cell invasive behaviour.
  • an inactive/inhibited or less active NMDAR signalling pathway is present in tumour cells expressing low GKAP, having low NMDAR pathway activity, and which are mostly non-invasive in nature.
  • GKAP acts as an effector of NMDAR signalling that is instrumental for the invasive tumour growth in both neuroendocrine and pancreatic ductal cancers, as well as the molecular mechanisms involved in these cellular responses. It has been established by the present inventors that GKAP governs invasive growth and treatment response to NMDAR inhibitors of PanNET via its pivotal role in regulating NMDAR pathway activity. It has further been acknowledged that downstream effectors FMRP and HSF-1 function along with GKAP to support invasiveness of PanNET and pancreatic ductal adenocarcinoma cancer cells.
  • transcriptome signatures that correlates with an invasive or non-invasive phenotype, being indicative of active vs. inactive/inhibited/less active NMDAR signalling, of pancreatic cancer, and in turn to a patient’s prognosis.
  • said transcriptome signatures were identified in tumours with low/inhibited NMDAR activity that significantly associate with favourable patient prognosis in several cancer types such as brain cancers (e.g. glial brain cancer), kidney cancers, skin cancer like uveal melanoma and blood cancer like acute myeloid leukaemia (AML).
  • brain cancers e.g. glial brain cancer
  • kidney cancers e.g. glial brain cancer
  • AML acute myeloid leukaemia
  • the present disclosure is not intended to be limited to the reported examples, and it is understood that the identified signatures can be used to determine cancer invasiveness as well as patient’s prognosis for other type of cancers such as breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract and/or thyroid cancer.
  • Those gene signatures have been identified in a mouse model of pancreatic cancer as four sets of gene products altered in their expression due to the genetic background of the tested subject and/or due to a treatment of the tested subject with a prototypical antagonist of the NMDA receptor, MK-801 (also known as Dizocilpine).
  • a so-called “strain signature” was identified, distinguishing gene products differently modulated in tumour samples from mouse strains having different genetic backgrounds (mouse strains B6 and C3H). It was later assessed, on the mouse model strain having the genetic background providing the most invasive tumour progression (B6 mouse strain), that some gene products are modulated (i.e. up-/down-regulated or up-/down-expressed) depending on the treatment with MK-801 , defining a so-called“MK-801 treatment signature”.
  • a sub-signature has been identified, defining a so-called“NMDAR- pathway l0W gene signature”, in which gene products are oppositely expressed in B6 mice compared to C3H mice (having the genetic background providing the less invasive tumour progression), as well as compared to B6 mice treated with MK-801 .
  • Said gene signatures identified in mouse models have been later on validated on cancer samples from humans, thus confirming the results of the initial findings also in higher mammals.
  • TABLE 4 list of genes whose gene products are down-regulated in NMDAR- pathway l0W gene signature. Accordingly, in one aspect, the down-regulation of a plurality of genes or gene products selected from the genes listed in Table 1 and/or 3, and/or up-regulation of a plurality of genes or gene products selected from the genes listed in Table 2 and/or 4, correlates with a high activation of the NMDAR signalling pathway in the cancer cells and therefore with a poor prognosis, whereas up-regulation of a plurality of genes or gene products selected from the genes listed in Table 1 and/or 3, and/or down-regulation of a plurality of genes or gene products selected from the genes listed in Table 2 and/or 4 correlates with a low activation of the NMDAR signalling pathway in the cancer cells and therefore with a favourable prognosis.
  • the predictive methods of the present disclosure are valuable tools in predicting if a subject like a human cancer patient is likely to respond favourably to a treatment regimen, such as surgical intervention, chemotherapy with a given drug or drug combination, and/or radiation therapy, or whether long-term survival of the patient, following surgery and/or termination of chemotherapy or other treatment modalities, is likely.
  • a treatment regimen such as surgical intervention, chemotherapy with a given drug or drug combination, and/or radiation therapy, or whether long-term survival of the patient, following surgery and/or termination of chemotherapy or other treatment modalities, is likely.
  • the predictive methods of the present disclosure are useful for selecting a subject affected with a cancer for a therapy and/or in determining whether a subject like a human patient is likely to respond favourably to, or benefit from, a treatment regimen based on at least one of a plurality of active compounds, such as agonists or antagonists of:
  • GKAP/DLGAP1 refers to guanylate kinase-associated protein, also known as Disks large-associated protein 1 (DAP-1). Accession numbers corresponding to the human GKAP/DLGAP1 gene in Genbank are NM_004746.3, NM_001003809.2, NM_001242761.1 , NM_001242762.1 ,
  • NM_001308390.1 in total 9 transcript variants
  • accession numbers corresponding to the human GKAP proteins are NP_004737, NP_001295319, NP_001229692, NP_001003809, NP_001229693, NP_001229690,
  • DLGAP1 GKAP is a protein known to be highly enriched in synaptosomal preparations of the brain, and it has been shown to facilitate the assembly of the post synaptic density of neurons.
  • the term“DLGAP family genes” refers to DLGAP1 and other four structurally-related genes, including DLGAP2 (GenelD: 9228), DLGAP3 (GenelD: 58512), DLGAP4 (GenelD: 22839) and DLGAP5 (GenelD: 9787).
  • HSF1 refers to the heat shock transcription factor 1. Accession number corresponding to the human HSF1 gene in Genbank is NM_005526, and accession number corresponding to the human HSF1 protein is
  • HSF1 This protein is encoded by the gene HSF1 (GenelD: 3297).
  • the product of this gene is a transcription factor that is rapidly induced after temperature stress and binds heat shock promoter elements.
  • HSF1 is the primary mediator of transcriptional responses to proteotoxic stress with important roles in non-stress regulation such as development and metabolism.
  • the HSF1 protein regulates the heat shock response (HSR) pathway in humans by acting as the major transcription factor for heat shock proteins, ensuring proper folding and distribution of proteins within cells. This pathway is induced by not only temperature stress, but also by a variety of other stressors such as hypoxic conditions and exposure to contaminants.
  • HSF1 transactivates genes for many cytoprotective proteins involved in heat shock, DNA damage repair, and metabolism.
  • FMRP refers to the fragile X mental retardation protein. Accession numbers corresponding to the human FMRP gene (FMR1 ) in Genbank are NM_001 185076, NMJD02024, NM_001 185082, NM_001 185075,
  • NM_001 185081 and accession numbers corresponding to the human FMRP proteins are NP_001 172005, NP_002015, NP_001 17201 1 , NP_001 172004, NP_001 172010.
  • This protein is encoded by the gene FMR1 (GenelD: 2332).
  • FMRP most commonly found in the brain, is essential for normal cognitive development and female reproductive function. FMRP has been suggested to play roles in nucleocytoplasmic shuttling of mRNA, dendritic mRNA localization, and synaptic protein synthesis.
  • FMRP is an RNA- binding protein which associates with polyribosomes and has been shown to inhibit translation of mRNA. Mutations of this gene can lead to fragile X syndrome, intellectual disability, premature ovarian failure, autism, Parkinson's disease, developmental delays and other cognitive deficits.
  • the gene product is a polypeptide.
  • the quantity of protein may be measured by semi-quantitative Western blots, enzyme-labelled and mediated immunoassays, such as ELISAs, biotin/avidin type assays, radioimmunoassay, Immunoelectrophoresis or immunoprecipitation or by protein or antibody arrays.
  • the protein expression level may be assessed by immunohistochemistry on a tissue section of the sample (e.g. frozen or formalin-fixed paraffin embedded material).
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the quantity of protein is measured by immunohistochemistry or semi-quantitative western-blot.
  • the gene product is mRNA.
  • Methods for determining the quantity of mRNA are well known in the art.
  • the nucleic acid contained in the sample e.g., cell or tissue prepared from the patient
  • the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR).
  • hybridization e. g., Northern blot analysis
  • amplification e.g., RT-PCR
  • quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.
  • primer pairs were designed in order to overlap an intron, so as to distinguish cDNA amplification from putative genomic contamination.
  • Other primers may be easily designed by the skilled person.
  • Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
  • LCR ligase chain reaction
  • TMA transcription- mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • the quantity of mRNA is measured by quantitative or semi-quantitative RT-PCR or by real-time quantitative or semi-quantitative RT-PCR or by transcriptome approaches.
  • RNA-sequencing RNA-sequencing (RNAS_seq) is commonly used as well, and it is herein preferred.
  • the expression level and/or the transcription level and/or the activity of a plurality of genes or gene products is determined, said plurality of genes or gene products comprising at least 2, or at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30, or at least 35, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100, or at least 150, or at least 200, or at least 250, or at least 300 prognostic genes or gene products.
  • said plurality of genes or gene products comprises or consists of the totality of the 330 genes or gene products listed in Tables 1 and 2.
  • said plurality of genes or gene products comprises or consists of the totality of the 148 genes or gene products listed in Tables 3 and 4.
  • said plurality of genes or gene products comprises or consists of the following genes or gene products:
  • the modulation of the expression level and/or the transcription level and/or the activity of genes or gene products of the above comprises increasing the expression level and/or the transcription level and/or the activity of genes or gene products whose up-regulation or over-expression is associated with a good prognosis, and in a preferred aspect the genes or gene products are selected from a group comprising or consisting of the genes or gene products listed in Tables 1 and/or 3.
  • the genes or gene products whose up-regulation or over- expression is associated with a good prognosis, and for which increase in the expression level and/or the transcription level and/or the activity is sought comprise or consist of the following:
  • the modulation of the expression level and/or the transcription level and/or the activity of genes or gene products comprises decreasing the expression level and/or the transcription level and/or the activity of genes or gene products whose down-regulation or down-expression is associated with a good prognosis, and in a preferred aspect the genes or gene products are selected from a group comprising or consisting of the genes or gene products listed in Tables 2 and/or 4.
  • the genes or gene products whose down-regulation or down- expression is associated with a good prognosis, and for which decrease in the expression level and/or the transcription level and/or the activity is sought comprise or consist of the following:
  • the subject-matter described in the present specification further provides a method for improving the prognosis for a cancer patient being diagnosed to have a poor prognosis, comprising modulating the expression level and/or the transcription level and/or the activity of:
  • said modulation is carried out by administering to a cancer patient: a. an inhibitor of at least one gene, or gene product derived therefrom, listed in Tables 2 and/or 4; and/or
  • an inhibitor of the HSF1 gene or of gene product(s) derived therefrom f. an inhibitor of the HSF1 gene or of gene product(s) derived therefrom; and/or g. an inhibitor of the FMR1 gene or of gene product(s) derived therefrom.
  • the method can be used to improve the prognosis of a patient being affected by a cancer selected from a list comprising blood cancer such as acute myeloid leukaemia, breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma such as uveal melanoma, and brain cancer such as glial brain cancer.
  • blood cancer such as acute myeloid leukaemia, breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma such as uveal melanoma, and brain cancer such as glial brain cancer.
  • compositions for use in the treatment of a cancer in a patient being diagnosed to have a poor prognosis comprising:
  • an inhibitor of the NMDA receptor c. an inhibitor of the NMDA receptor; and/or d. an inhibitor of a gene product member of the NMDA receptor signalling pathway; and/or
  • an inhibitor of the FMR1 gene or of gene product(s) derived therefrom.
  • Said inhibitors or activators can be selected from a non-exhaustive list comprising a nucleic acid molecule, a polypeptide, a fusion protein, an antibody as well as a derivative or fragment(s) thereof.
  • said inhibitor is selected from a group including but not limited to MK801 , memantine, Ifenprodil, ketamine and Ro 25-6981.
  • the pharmaceutical composition can be used to improve the prognosis of a patient being affected by a cancer selected from a list comprising blood cancer such as acute myeloid leukaemia, breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma such as uveal melanoma, and brain cancer such as glial brain cancer.
  • blood cancer such as acute myeloid leukaemia, breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma such as uveal melanoma, and brain cancer such as glial brain cancer.
  • said plurality of genes or gene products comprises at least 2, or at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30, or at least 35, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100, or at least 150, or at least 200, or at least 250, or at least 300 of said genes or gene products.
  • said plurality of genes or gene products comprises or consists of the totality of the 330 genes or gene products listed in Tables 1 and 2.
  • said plurality of genes or gene products comprises or consists of the totality of the 148 genes or gene products listed in Tables 3 and 4.
  • said plurality of genes or gene products comprises or consists of the following genes or gene products:
  • compositions may, optionally and additionally, comprise a pharmaceutically acceptable carrier, excipient and/or diluent.
  • “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, that are physiologically compatible.
  • suitable pharmaceutical carriers include sodium chloride solutions, phosphate buffered sodium chloride solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, organic solvents and so forth.
  • the pharmaceutically acceptable carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as e.g.
  • polypeptides e.g., polyarginine or tripeptides
  • proteins such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyvinylpyrrolidone
  • amino acids such as glycine, glutamic acid, aspartic acid, or arginine
  • monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins
  • chelating agents such as EDTA
  • sugar alcohols such as mannitol or sorbitol
  • counterions such as sodium
  • non-ionic surfactants such as polysorbates, poloxamers, or PEG.
  • a “therapeutically effective dose” refers to that amount of active ingredient, for example a nucleic acid molecule, a polypeptide, a fusion protein, an antibody as well as a derivative or fragment(s) thereof according to the present disclosure, which ameliorates the signs or symptoms of the disease or prevent progression thereof; as would be understood in the medical arts, cure, although desired, is not required.
  • the therapeutically effective dose of the pharmaceutical agents described herein can be estimated initially by in vitro tests, such as cell culture assays, followed by assay in model animals, usually mice, rats, rabbits, dogs, or pigs. The animal model can also be used to determine an initial preferred concentration range and route of administration.
  • inhibitors and/or activators can be various and diverse in nature, and are particularly intended for use in the treatment of human cancer patients having a poor prognosis.
  • Said treatments can include for instance gene therapy and/or treatment with therapeutic molecules including but not limited to antibodies, small molecules and nucleic acids such as antisense molecules.
  • the inhibitor of the invention is an antisense molecule that interferes with the activity or expression of the nucleic acid sequence (e.g. RNA) of the invention by hybridizing to the nucleic acid sequence of the corresponding nucleic acid sequence or to a variant or fragment thereof.
  • the nucleic acid sequence e.g. RNA
  • antisense molecules include, e.g., miRNA, siRNA, piRNA, snRNA, sh RNA or a modified antisense molecule such as GapmeRs.
  • microRNA refers to endogenous or artificial non-coding RNAs that are capable of regulating gene expression. It is believed that miRNAs function via RNA interference.
  • siRNA and“short interfering RNA” are interchangeable and refer to single- stranded or double-stranded RNA molecules that are capable of inducing RNA interference. siRNA molecules typically have a duplex region that is between 18 and 30 base pairs in length.
  • PiRNA and“Piwi-interacting RNA” are interchangeable and refer to a class of small RNAs involved in gene silencing. PiRNA molecules typically are between 26 and 31 nucleotides in length.
  • RNA small nuclear RNA
  • RNA splicing and regulation of transcription factors.
  • subclass of small nucleolar RNAs The subclass of small nucleolar RNAs
  • RNAs is also included.
  • the term is also intended to include artificial snRNAs, such as antisense derivatives of snRNAs comprising antisense sequences directed against one or more nucleic acid sequence of the invention, or to a variant or fragment thereof.
  • shRNA refers to a nucleic acid molecule comprising at least two complementary portions hybridized or capable of hybridizing to form a duplex structure sufficiently long to mediate RNAi (typically between 15-29 nucleotides in length), and at least one single-stranded portion, typically between approximately 1 and 10 nucleotides in length that forms a loop connecting the ends of the two sequences that form the duplex.
  • a GapmeR is a chimeric antisense oligonucleotide that contains a central block of deoxynucleotide monomers sufficiently long to induce RNase H cleavage.
  • the GapmeRs of the invention are directed against one or more nucleic acid sequence of the invention, or to a variant or fragment thereof.
  • an “antibody” refers to an intact immunoglobulin, or to an antigen-binding portion thereof that competes with the intact antibody for specific binding to a molecular species, e.g., a polypeptide involved in (i.e. being part of) the NMDAR signalling pathway.
  • Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding portions include, inter alia, Fab, Fab', F(ab')2 , Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
  • CDR complementarity determining region
  • a Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab')2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consists of the VH and CH1 domains; a Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment consists of a VH domain.
  • bind specifically and “specific binding” as used herein it is meant the ability of the antibody to bind to a first molecular species in preference to binding to other molecular species with which the antibody and first molecular species are admixed.
  • An antibody is said specifically to "recognize” a first molecular species when it can bind specifically to that first molecular species.
  • a single-chain antibody (scFv) is an antibody in which VL and VH regions are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites.
  • One or more CDRs may be incorporated into a molecule either covalently or non-covalently to make it an immunoadhesin.
  • An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) non-covalently.
  • the CDRs permit the immunoadhesin to specifically bind to a particular antigen of interest.
  • a chimeric antibody is an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies.
  • An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a "bispecific" or “bifunctional” antibody has two different binding sites.
  • an “isolated antibody” is an antibody that 1 ) is not associated with naturally- associated components, including other naturally-associated antibodies, that accompany it in its native state, 2) is free of other proteins from the same species, 3) is expressed by a cell from a different species, or 4) does not occur in nature. It is known that purified proteins, including purified antibodies, may be stabilized with non-naturally associated components.
  • the non-naturally-associated component may be a protein, such as albumin (e.g., BSA) or a chemical such as polyethylene glycol (PEG).
  • a “neutralizing antibody” or “an inhibitory antibody” is an antibody that inhibits the activity of a polypeptide or blocks the binding of a polypeptide to a ligand that normally binds to it.
  • An “activating antibody” is an antibody that increases the activity of a polypeptide.
  • epitopic determinants includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An antibody is said to specifically bind an antigen when the dissociation constant is less than 1 ⁇ M, preferably less than 100 nM and most preferably less than 10 nM.
  • nucleic acid molecule refers to a polymeric form of nucleotides and includes both sense and antisense strands of RNA (e.g. mRNA, siRNA, shRNA), cDNA, genomic DNA, and synthetic forms and mixed polymers of the above.
  • a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide.
  • a “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.”
  • the term “nucleic acid molecule” usually refers to a molecule of at least 10 bases in length, unless otherwise specified. The term includes single and double stranded forms of DNA.
  • a polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
  • nucleic acid molecules may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.)
  • the term "nucleic acid molecule" also includes any topological conformation, including single- stranded, double-
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • Antisense nucleic acid compositions, or vectors that drive expression of an antisense nucleic acid are administered to downregulate transcription and/or translation of a gene product in circumstances in which e.g. excessive production, or production of aberrant protein, is the pathophysiologic basis of disease.
  • Antisense compositions useful in therapy can have a sequence that is complementary to coding or to noncoding regions of a gene or gene product deriving therefrom. For example, oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and + 10 from the start site, are preferred.
  • Catalytic antisense compositions, such as ribozymes, that are capable of sequence-specific hybridization to gene transcripts are also useful in therapy.
  • the antisense is an oligonucleotide (e.g. an antisense molecule as described herein) having a sequence designed to hybridize with a nucleic acid sequence of a gene, or gene product such as an mRNA deriving therefrom, selected from a list including:
  • nucleic acid as well as a fragment, an allelic variant, a homolog and/or a substantially similar or hybridizing nucleic acid of the foregoing.
  • kits for in vitro analysis aimed at determining the prognosis of a patient having a cancer, said cancer being altered in the NMDAR signalling pathway, said kit comprising a reagent that selectively interacts with one or more of:
  • the kit of the invention may also comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the label or package insert may comprise instructions for use thereof. Instructions included may be affixed to packaging material or may be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and
  • the cancer can be selected from a list comprising blood cancer such as acute myeloid leukaemia, breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma such as uveal melanoma, and brain cancer such as glial brain cancer.
  • blood cancer such as acute myeloid leukaemia, breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma such as uveal melanoma, and brain cancer such as glial brain cancer.
  • Another aspect of the subject matter described in the present specification relates to a method of treatment of a cancer in a patient being diagnosed to have a poor prognosis, comprising administering:
  • an inhibitor of the FMR1 gene or of gene product(s) derived therefrom.
  • Said inhibitors or activators can be selected from a non-exhaustive list comprising a nucleic acid molecule, a polypeptide, a fusion protein, an antibody as well as a derivative, variant or fragment(s) thereof, as described herein.
  • said inhibitor is selected from a group including but not limited to MK801 , memantine, Ifenprodil, ketamine and Ro 25-6981.
  • the method of treatment described above follows the method for determining the prognosis for survival of a patient having a cancer of the invention.
  • a further object of the subject-matter described in this disclosure relates to the use of a pharmaceutical composition of the invention in the preparation of a medicament for the treatment of a cancer in a patient being diagnosed to have a poor prognosis.
  • Administration refers to contact of an effective amount of an inhibitor or activator of the invention, to the patient.
  • a composition comprising an inhibitor or activator that is in the form of a miRNA, siRNA, piRNA, hnRNA, snRNA, esiRNA, shRNA, or antisense oligonucleotide are well known in the art.
  • routes of administration already in use for nucleic acid therapeutics, along with formulations in current use, provide preferred routes of administration and formulation for the nucleic acids described herein.
  • Nucleic acid compositions can be administered by a number of routes including, but not limited to: oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, intra- or transcranial or rectal means. Nucleic acids can also be administered via gene delivery vectors or nanoparticules. Such administration routes and appropriate formulations are generally known to those of skill in the art.
  • a gene delivery vector preferably in the form of a plasmid or a vector, comprises one or more nucleic acid(s) encoding an inbitor or activator of the present invention.
  • a "vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e. g.,
  • yeast plasmids such as the 2* plasmid or derivatives thereof
  • vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells
  • vectors derived from combinations of plasmids and phage DNAs such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • viral vectors are used for delivering nucleic acid to cells in vitro or in vivo.
  • Non-limiting examples are vectors based on Herpes Viruses, Pox- viruses, Adeno- associated virus, Lentivirus, and others. In principle all of them are suited to deliver the expression cassette comprising an expressible nucleic acid molecule that codes for an inbitor or activator of the present invention.
  • said viral vector is an adenoviral vector, preferably a replication competent adenovirus.
  • compositions comprising an inhibitor or activator that is in the form of a polypeptide, a fusion protein, an antibody as well as a derivative, variant or fragment(s) thereof, as described herein are also well known in the art.
  • Administration of the formulations may be accomplished by any acceptable method which allows the inhibitor or activator of the invention to reach its target.
  • the particular mode selected will depend of course, upon factors such as the particular formulation, the severity of the state of the subject being treated, and the dosage required for therapeutic efficacy.
  • the actual effective amounts of drug can vary according to the specific drug or combination thereof being utilized, the particular composition formulated, the mode of administration, and the age, weight, condition of the patient, and severity of the symptoms or condition being treated.
  • any acceptable method known to one of ordinary skill in the art may be used to administer a formulation to the subject.
  • the administration may be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the cancer being treated.
  • Injections can be e.g., intravenous, intradermal, subcutaneous, intra- or
  • Implantation includes inserting implantable drug delivery systems, e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol matrixes, polymeric systems, e.g., matrix erosion and/or diffusion systems and non-polymeric systems, e.g., compressed, fused, or partially-fused pellets.
  • implantable drug delivery systems e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol matrixes, polymeric systems, e.g., matrix erosion and/or diffusion systems and non-polymeric systems, e.g., compressed, fused, or partially-fused pellets.
  • Inhalation includes administering the composition with an aerosol in an inhaler, either alone or attached to a carrier that can be absorbed. For systemic administration, it may be preferred that the composition is
  • the antisense nucleic acid compositions, or vectors or plasmids that drive expression of an inhibitor or activator of the invention are provided in a manner which enables tissue-specific uptake of the nucleic acid delivery system.
  • Techniques include using tissue or organ localizing devices, such as wound dressings or transdermal delivery systems, using invasive devices such as vascular or urinary catheters, and using interventional devices such as stents having drug delivery capability and
  • the formulations may be delivered using a bio-erodible implant by way of diffusion or by degradation of the polymeric matrix.
  • the administration of the formulation may be designed so as to result in sequential exposures to an inhibitor or activator of the invention, or the pharmaceutical composition comprising the same, over a certain time period, for example, hours, days, weeks, months or years. This may be accomplished, for example, by repeated administrations of a formulation or by a sustained or controlled release delivery system in which the inhibitor or activator of the invention or the
  • compositions comprising the same, is delivered over a prolonged period without repeated administrations.
  • Administration of the formulations using such a delivery system may be, for example, by oral dosage forms, bolus injections, transdermal patches or subcutaneous implants. Maintaining a
  • substantially constant concentration of the composition may be preferred in some cases.
  • delivery systems suitable include, but are not limited to, time-release, delayed release, sustained release, or controlled release delivery systems. Such systems may avoid repeated administrations in many cases, increasing
  • release delivery systems are available and known to those of ordinary skill in the art. They include, for example, polymer-based systems such as polylactic and/or polyglycolic acids, polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides,
  • polyorthoesters polyhydroxybutyric acid, and/or combinations of these.
  • Microcapsules of the foregoing polymers containing nucleic acids are described in, for example, U.S. Patent No. 5,075, 109.
  • Other examples include nonpolymer systems that are lipid-based including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; liposome-based systems; phospholipid based-systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants.
  • Specific examples include, but are not limited to, erosional systems in which the inhibitor or activator is contained in a formulation within a matrix (for example, as described in U.S. Patent Nos. 4,452,775, 4,675, 189, 5,736,152, 4,667,013, 4,748,034 and
  • the formulation may be as, for example, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems.
  • the system may allow sustained or controlled release of the composition to occur, for example, through control of the diffusion or erosion/degradation rate of the formulation containing the inhibitor or activator of the invention.
  • a pump-based hardware delivery system may be used for delivery. Examples
  • Dlgap 1/GKAP is the most differentially expressed candidate modifier gene when comparing the invasive B6 and non-invasive C3H backgrounds
  • Cancer cells from the invasive B6 background have higher NMDAR pathway activity than those from the non-invasive C3H background
  • B6 cancer cells (* TC-B6) were found to be more invasive than C3H cancer cells (bTO03H) ( Figure 1 E). Notably, under flow conditions glutamate secretion was significantly increased in * TC-B6 compared to * TC-C3H ( Figure 1 F) compared to static conditions.
  • GKAP expression is associated with differential NMDAR activity and sensitivity to pharmacological inhibition in tumours
  • the NMDAR signalling pathway includes the NMDAR subunits GluN1 (Grinl) and GluN2b (Grin2b), the key scaffold proteins PSD-95 (Dlg4) and GKAP (Dlgapl), and the principal glutamate transporters vGlutl (Sid 7a8) and vGlut2 (Sid 7a7).
  • GKAP has nine protein-coding splice variants, of which three were differentially expressed in B6 vs C3H PanNETs ( Figure 1 1 B) (the remaining six variants were all below the detection threshold). Accordingly, a shRNA knockdown design that targeted all three of these GKAP isoforms was selected.
  • the GKAP-knockdown cells showed decreased GluN2b phosphorylation, indicative of reduced NMDAR signalling activity (Figure 4A).
  • GKAP-knockdown cells exhibited a significantly reduced response to the NMDA ligand (Figure 4B).
  • ⁇ TC-3 invasiveness under flow conditions was also decreased by the knockdown ( Figure 4C).
  • the knockdown of GKAP mRNA functionally phenocopied the C3H phenotype, in relation to NMDAR activity and cancer cell invasiveness.
  • NMDAR pathway activity modulates several regulators of the translational machinery
  • FAM107a family with sequence similarity 107, also known as DRR1
  • DRR1 sequence similarity 107
  • 1 1 .72
  • the inventors aimed at identifying a common (core) gene expression profile for the NMDAR pathway-low phenotype.
  • core common (core) gene expression profile for the NMDAR pathway-low phenotype.
  • the inventors identified a total of 148 genes, and named this signature as the “NMDAR-pathway l0W gene/NMDAR pathway-low signature” ( Figure 7D; Tables 3 and 4).
  • NMDAR-pathway low gene signature identified “Neurogenesis and Synaptogenesis” to be one of the major pathways involving these genes ( Figure 1 1 A), consistent with the interpretation that these genes are regulated by NMDAR signalling.
  • one of the major phenotypes of B6 MK801 -treated tumours is decreased proliferation hence low tumour burden, in contrast to C3H tumours ( Figure 7A); therefore, applying these selection criteria allows us to exclude common genes associated with proliferation, and focus on genes specific reflecting NMDAR pathway activation.
  • MK801 treatment was further evaluated in experimental therapeutic trials performed in the PDAC GEMM (LSL- KrasG12D; p53LSLR172H; p48Cre) using the pharmacological inhibitor MK801.
  • a survival benefit was also observed using a clinically approved, albeit less potent NMDAR inhibitor, memantine (Figure 8A).
  • Transcriptome signatures identified in mPanNETs can predict prognosis in various human cancer types
  • a remarkable and significant survival benefit was associated with PDAC patients whose tumours correlated with the “MK801 -treatment signature” - as if tumours from these patients had been treated with MK801 - compared to the rest of the patients ( Figure 8B); moreover, lower grade tumours (G1 , G2) showed a more pronounced association with the MK801 treatment signature compared to higher grade tumours (G3, G4)( Figure 12A). It was found that the“MK801 treatment signature” is a significant, independent prognostic factor, even surpassing the predictive power of T and N stages (Figure 8C).
  • this “MK801 treatment signature” was also significantly associated with favourable prognosis in patients from several other cancer types, including glial brain cancers, kidney cancers, uveal melanoma, and acute myeloid leukaemia (Figure 8D).
  • glial brain cancers including glial brain cancers, kidney cancers, uveal melanoma, and acute myeloid leukaemia ( Figure 8D).
  • glial brain cancers low grade glioma tumours were significantly more correlated with the MK801 treatment/pathway-low signature when compared to advanced (more invasive and aggressive) glioblastoma tumours (Figure 8E), suggestive of the latter’s dependence on NMDAR pathway activity to drive the highly malignant phenotype.

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Abstract

A method for determining the prognosis for survival of a patient having cancer is disclosed, said cancer being altered (i.e. highly activated or alternatively inactive/inhibited) in the NMDAR signalling pathway, said method comprising determining the expression levels of a plurality of gene products in a sample from a cancer patient relative to a control, wherein the differential expression of said plurality of gene products relative to a control is indicative of cancer aggressiveness and of the patient's prognosis. A method for improving the prognosis for a cancer patient being diagnosed to have a poor prognosis, as well as pharmaceutical compositions for that purpose, are also disclosed, said method comprising modulating the level or the activity of the identified gene products and/or a member of the NMDA signalling pathways and/or a DLGAP family gene products and/or a HSF1 gene product and/or a FMR1 gene product.

Description

Method for determining cancer invasiveness and patient prognosis
Technical Field
The subject matter described in the present disclosure lies in the field of molecular oncology. More particularly, the subject-matter described in the present disclosure relates to a method for determining and/or improving the prognosis for survival of a cancer patient, said method being based on assessment of the activation profile of the NMDAR signalling pathway that stimulates malignant tumour growth and impacts a cancer patient’s prognosis. Background of the Invention
While distinct oncogenic “driver” genes are widely appreciated to be instrumental in cancer progression, the contribution of “modifier genes” has been relatively less well studied. Modifier genes can modulate the penetrance of specific driver oncogenes, exerting either protective or detrimental effects and affecting therapeutic outcome. Numerous studies employing quantitative trait locus (QTL) mapping in mouse and genome-wide association analyses (GWAS) in human have identified potential genetic modifier loci; however, few of these genetic modifiers have been validated mechanistically. Elucidating how genetic polymorphisms affect tumourigenesis at the molecular level is an important step towards appreciating individual variation in prognosis, and in implementing personalized cancer therapies.
The RIP1 Tag2 transgenic mouse model of pancreatic neuroendocrine tumour (PanNET) recapitulates the multi-stage nature of human cancer progression. As such, it has proved to be a valuable research tool for elucidating mechanisms of tumour development and malignant progression to invasive and metastatic states. Interestingly, varying degrees of tumour invasiveness are observed at end stage depending on the genetic background in which the same transgene integration is resident, despite expressing similar levels of the driving oncoprotein (SV40 T- antigen) under control of the Rat Insulin Promoter (RIP). In particular, the C57BI/6 (B6) background gives rise to highly invasive carcinomas, whereas mice in the C3HeB/Fe (C3H) background primarily develop well-defined, non-invasive islet tumours (Chun et al., 2010). Therefore, the RIP1Tag2 model may phenocopy a facet of the complexity of cancer progression in patients, where the same oncogenetic events can lead to varying outcomes in different patient populations.
Starting from the observation of the influence of the genetic background on cancer invasiveness in the RIP1Tag2 mouse model, the inventors took advantage of a previous report which identified quantitative trait loci associated with an invasive cancer phenotype in mice. Through a classical linkage analysis, a QTL was detected on mouse chromosome 17 that is highly associated with an invasive phenotype of pancreatic neuroendocrine tumour. As such, this locus was postulated to be a candidate“modifier locus” for mouse PanNET progression (Chun et al., 2010).
The identified region harbours more than 50 genes, having no polymorphic differences in their coding regions of these genes. Seven genes were identified as the most differentially expressed in mPanNETs arising in the B6 versus the C3H genetic strain backgrounds, and implicated as potential invasion modifier candidates. The inventors focused later on their attention on dlgap1/GKAP, a key adaptor protein of the glutamate-activated N-methyl-D-aspartate receptors (NMDA receptor, also known as GluNR) in the central nervous system. NMDAR is an important neuronal receptor involved in learning and memory, regulating synaptic plasticity in the central nervous system (Abbott and Nelson, 2000). GKAP clusters and potentiates NMDAR activity via its interaction with the upstream scaffold protein PSD-95, which directly binds the NMDAR; moreover, it organizes and stabilizes a variety of NMDAR scaffold complexes to transmit signals downstream. It was previously established that NMDAR signalling is active and promotes invasive tumour growth in the pro-invasive B6 background of this mouse model of PanNET, where GKAP is highly expressed (Li and Hanahan, 2013). Nonetheless, how exactly the genetic background of an individual could impact cancer invasiveness and progression, as well as possible gene signatures influencing this complex process, is a controversial argument yet under debate. Methods and compounds to ascertain and modulate cancer invasiveness so to promptly intervene and improve patients’ prognosis are still urgently needed. Therefore, there is a great need for the identification of prognostic markers that can accurately distinguish tumours associated with good prognosis including low probability of metastasis, late disease progression, decreased disease recurrence or increased patient survival, from the others. Moreover, since different signalling pathways may be dominant in different patients, it is only by identifying patients whose tumours have activated specific molecular pathways using such markers, that the practitioner can predict the patient's prognosis and can effectively target the individuals who would most likely benefit from therapy or who need a more intensive monitoring.
Summary of Invention
According to one object of the subject-matter described in this disclosure, it is provided herein a method for determining the prognosis for survival of a patient having a cancer, said cancer being altered (i.e. highly activated or alternatively inactive/inhibited) in the NMDAR signalling pathway, said method comprising a step of determining the transcription level and/or expression level and/or the activity of a plurality of genes or gene products selected from genes listed in Tables 1 to 4 in a sample from said cancer patient relative to a control, wherein the differential transcription and/or expression and/or activity of said plurality of genes or gene products relative to a control is indicative of cancer aggressiveness and therefore of the patient’s prognosis.
Another object of the subject-matter described in this disclosure relates to a method for improving the prognosis for a cancer patient being diagnosed to have a poor prognosis, comprising modulating the transcription level and/or the expression level and/or the activity of:
- a plurality of genes or gene products, or at least one of said genes or gene products, listed in Tables 1 to 4; and/or
- a gene or gene product member of the NMDA receptor signalling pathway; and/or
- a gene or gene product member of the DLGAP family of genes (including DLGAP1 , DLGAP2, DLGAP3, DLGAP4 and DLGAP5); and/or
- a HSF1 gene or gene product; and/or
- a FMR1 gene or gene product.
In one aspect, said modulating is carried out by administering to said cancer patient:
a. (an) inhibitor(s) of at least one gene, or gene product derived therefrom, listed in Tables 2 and/or 4; and/or
b. at least one gene, or gene product derived therefrom, listed in Tables 1 and/or 3, or (an) activator(s) thereof; and/or
c. (an) inhibitor(s) of the NMDA receptor; and/or
d. (an) inhibitor(s) of a gene product member of the NMDA receptor signalling pathway; and/or
e. (an) inhibitor(s) of a member of the DLGAP family of genes, or of gene product(s) derived therefrom; and/or
f. (an) inhibitor(s) of the HSF1 gene, or of gene product(s) derived therefrom; and/or
g. (an) inhibitor(s) of the FMR1 gene, or of gene product(s) derived therefrom.
A further object of the subject-matter described in this disclosure relates to a pharmaceutical composition for use in the treatment of a cancer in a patient being diagnosed to have a poor prognosis, comprising: a. (an) inhibitor(s) of at least one gene, or gene product derived therefrom, listed in Tables 2 and/or 4; and/or
b. at least one gene, or gene product derived therefrom, listed in Tables 1 and/or 3, or (an) activator(s) thereof; and/or
c. (an) inhibitor(s) of the NMDA receptor; and/or
d. (an) inhibitor(s) of a gene product member of the NMDA receptor signalling pathway; and/or
e. (an) inhibitor(s) of a member of the DLGAP family of genes, or of gene product(s) derived therefrom; and/or
f. (an) inhibitor(s) of the HSF1 gene, or of gene product(s) derived therefrom; and/or
g. (an) inhibitor(s) of the FMR1 gene, or of gene product(s) derived therefrom.
Still a further object of the subject-matter described in this disclosure relates to a kit for in vitro analysis aimed at determining the prognosis of a patient having a cancer, said cancer being altered in the NMDAR signalling pathway, said kit comprising a reagent that selectively interacts with one or more of:
(a) at least one of a plurality of gene products of Tables 1 to 4; and/or
(b) a nucleic acid molecule having at least 95% sequence identity to a gene product derived from (a); and/or
(c) a polypeptide encoded by a nucleic acid molecule of (b); and/or
(d) a polypeptide comprising an amino acid sequence with at least 95% sequence identity to a polypeptide of (b).
The above and other objects, features and advantages of the herein presented subject matter will become more apparent from a study of the following description with reference to the attached figures. Brief description of the Figures
Figure 1 shows the differential GKAP expression between the C57/BI6 and C3Heb/Fe genetic backgrounds associated with a differential NMDAR pathway activity in vitro:
(A) qRT-PCR of GKAP mRNA in mPanNET tumour-derived cancer cell lines (· TC- B6 and * TC-C3H) and primary tumours that arose in RIP1-Tag2 transgenic mice inbred into the B6 and C3H backgrounds, respectively (upper panel). Western blot for GKAP protein expression (lower panel). Mean with SEM. *: p<0.05, **: p<0.01 (n=3 individual tumours/genetic background; n=3 independent RNA extraction/cell line);
(B) qRT-PCR analysis of FACS-sorted cell types from primary tumours derived from B6 mice. Cells were sorted from pools of multiple PanNETs isolated from 2 mice. Mean with SEM. One-way ANOVA, Dunnett multiple comparisons test was used when cancer cells were compared with all other populations (p<0.0001 in all comparisons);
(C) Among the multiple SNPs associated with the GKAP gene and its flanking sequences that distinguished C3H mice from B6 mice, a transcription factor prediction algorithm identified only one SNP that matched with a potential transcription factor binding site (upper panel). This site in the B6 version of the GKAP gene putatively binds to HSF1 (p<0.004). ChIP-qPCR for the GKAP SNP site after HSF1 immunoprecipitation (lower panel). The * maj (· globin, Hbb-b1 ) promoter region was used as negative control, as it lacks a binding site for HSF1. Mann-Whitney test, *: p=0.02, (n=4, 2 batches of cell lysates per cell line, and 2 qPCR/batch);
(D) Western blot for GKAP expression after HSF1 knockdown. HSF1 and GKAP protein expression levels were normalized to GAPDH and siRNA-control (Thermo Fisher Scientific, #4390771 , Assay ID s67870, s67871), the results of which are shown in the figure panels (n=3 independent experiments);
(E) · TC-B6 cancer cells were more invasive than · TC-C3H, especially under flow conditions wherein glutamate-stimulated NMDAR signalling is activated. Mean ± SEM. Two-way ANOVA, Bonferroni multiple comparisons test, n.s: not significant; **: p=0.0019; ***: p<0.001. (n=4 independent assays for static condition; n=6-9 for flow condition);
(F) Underflow conditions, · TC-B6 cells secreted more glutamate compared to · TC- C3H cells. Mean ± SEM. Two-way ANOVA, Bonferroni multiple comparisons test, ***: p<0.001 , ****: p<0.0001 , n.s.: not significant. (n=3 invasion assay devices/condition/cell line);
Figure 2 shows that the intracellular calcium responses and electrophysiology reveals functional NMDAR in bTOBQ but not in bT0-03H cells:
(A) Oregon Green-labeled calcium indicator BAPTA-AM was applied to · TC-B6 and - TC-C3H cancer cells bathed in a Mg-free Ringer solution; puffing an NMDA solution (1 mM, 1s, through perfusion pipette at left) induced calcium influx into the cells, thereby producing an increased fluorescence signal (· F) compared to the background fluorescence signal (F). In (i), the top panel shows · TC-B6 in phase- contrast, whereas the lower panel shows a green-fluorescence signal overlaid with a phase-contrast image, (ii) Time-resolved fluorescence signals (sampling frequency / frame rate = 12.5 Hz), where each trace represents one recorded cell. The Y-axis indicates the change in fluorescence intensity;
(B) Using the fluorescence reporter assay in (A), the number of · TC cells with active NMDAR signaling was determined following“puff application” of 1 mM NMDA. The · F/F measurements refer to the normalized difference in each cell’s signal measured immediately before the application of agonist compared to the peak of the response after the puff. Approximately 34% of · TC-B6 cells showed active NMDAR signaling (dark green bars, · F/F>0); in sharp contrast, none of the * TC- C3H cells exhibited active NMDAR signaling detectable using the same method (brown bar, · F/F=0). For * TC-B6, 263 cells from 15 different regions of 3 independent culture dishes were recorded; 90 had active NMDAR. For * TC-C3H, 155 cells from 8 regions of 2 different dishes were analysed; Wilcoxon rank sum test, p< 4.24e-16;
Figure 3 shows that high GKAP expression is associated with increased NMDAR pathway activity in vivo:
(A) qRT-PCR evaluation for the NMDAR subunits GluN1 (Grinl), GluN2b (Grin2b), and the scaffold protein GKAP (Dlgapl) in PanNET tumours from the two genetic backgrounds. Mean ± SEM, Mann-Whitney test was used to compare the expression of each gene in the B6 and C3H tumours, *: p=0.0175. (qRT-PCR: n = 7 tumours/7 mice/background);
(B) Western blot confirmed that GKAP, but not GluN2b, was differentially expressed in PNETs from B6 and C3H. After normalization, the one-column t-test was used for comparison, hypothetical value = 1 , *: p=0.01 , n.s.: not significant. (n=4 tumours/4 mice);
C) IHC analysis of PanNET tumour tissue sections showed that GluN2b was similarly expressed in the B6 and C3H genetic backgrounds. However, its active form - p-GluN2b - was selectively detectable in B6 PanNETs. Images are representative of >50 tumours from >10 RIP1Tag2 mice/background;
(D) MK801 decreased tumour burden in the B6 but not in C3H RIP1Tag2 mice. Cohorts of 7-9 mice were used for control (saline treated) and MK801 treatment in each genetic background; Mann-Whitney test, **: p<0.01 ; n.s: Not significant;
Figure 4 shows that GKAP regulates cancer cell invasion through NMDAR activity and downstream effectors FMRP and HSF1 :
(A) Western blot: GKAP IDIgapl knockdown in pTC-3 decreased GluN2b phosphorylation under unstimulated cell culture conditions. The numbers below indicate levels of p-GluN2b and GKAP normalized to b-actin; (B) Fluorescence reporter assay was performed in * TC-3 shRNA control (Openbiosystem, #RHS6848) and GKAP (Openbiosystem, Clone ID: TRCN0000088935.) knockdown (KD) cells lines, comparing calcium transients induced by puffing an NMDA solution onto the cells. Clusters of cultured control « TC-3 cells (i) or GKAP-KD b"IΌ-3 cells (ii) were analyzed under a bright field microscope after a NMDA solution (1 mM, 1 s) was puffed through perfusion pipette at left (upper panels). NMDA caused large synchronous calcium transients in a group of Control-KD cells (left panels); in contrast, GKAP-KD cells do not show responses synchronized to the NMDA puff, but rather only small spontaneous, asynchronous transients (right panels). (* TC-3 shRNA control (Openbiosystem, #RHS6848) KD cells: 78 of 284 cells examined showed a response; · TC-3 shRNA GKAP (Openbiosystem, Clone ID: TRCN0000088935.) KD cells: 5 of 178 cells showed minor NMDA responses, p < 10_11 , Wilcoxon rank sum test.) Histogram of transient amplitude, denoting time-resolved fluorescence signals (sampling frequency / frame rate = 12.5 Hz);
(C) Invasion assay in control and GKAP-knockdown · TC-3 tumour cells. Two-way ANOVA, Bonferroni multiple comparisons test, **: p<0.01 ; n.s.: not significant (mean ± SEM, n=3 invasion assay devices/condition in one experiment; two independent experiments were performed with consistent results);
(D) IHC staining of FMRP in primary and liver-metastatic B6 PanNETs. Similarly- sized invasive vs non-invasive primary tumours from the same section were used for comparison (tumour borders marked by dash line in the representative images). Augmented FMRP expression was observed in a rare mouse that showed multiple metastatic lesions in the liver (indicated by the arrow heads, tumour borders marked by dashed line). Images shown are representative of an analysis of >50 PanNETs from >10 B6 RIP1 Tag2 mice, 1 section/mouse, and all staining was performed in the same experiment. (Magnified lesion is representative of >100 metastases from one liver); (E) FMRP knockdown (upper panel) decreased bT03 invasion in the flow-guided invasion assay (lower panel). The numbers below indicate levels of FMRP normalized to GAPDH. Unpaired t-test, *: p<0.05. **: p<0.01. FMRP #1 and #2 indicate two different siRNA constructs (Thermo Fisher Scientific, #4390771 , Assay ID s66176, s66177) used (mean ± SD, n=3 invasion assay devices/condition in one experiment; two independent experiments);
(F) Western blots comparing FMRP and p-HSF1 expression in bTϋ-3 cancer cells infected with control shRNA (Openbiosystem, #RHS6848) or shRNA-GKAP lentiviral vectors (Openbiosystem, Clone ID: TRCN0000088935.) (left panel). GAPDH was used as a loading control. Western blots comparing expression of p- GluN2b, FMRP and p-HSF1 in bTO-3 cancer cells treated with either vehicle or MK801 (right panel). The numbers below indicate levels of p-GluN2b, FMRP and p- HSF1 normalized to GAPDH (n=3);
Figure 5 shows that NMDAR signaling through GKAP promotes invasion in both mouse and human PDAC cell lines:
(A) The high-GKAP-expressing mPDAC-4361 line had high p-GluN2b, whereas the low-GKAP-expressing mPDAC-2261 line had low p-GluN2b, as revealed by Western blot analysis (left panels). The numbers indicate quantification (n=3). Line 4361 was significantly more invasive than line 2261 , especially under flow condition (right panel). Two way ANOVA, Bonferroni’s multiple comparisons test (right panel), ****: p<0.0001 , *: p<0.05. (MeaniSD, n=3 invasion assay devices/condition/cell line in one experiment; two independent experiments);
(B) The GKAPh'9h mPDAC-4361 was also more sensitive to MK801 inhibition of invasion. The data were normalized to each corresponding“control” in the same static/flow conditions. Two way ANOVA, Bonferroni’s multiple comparisons test, n.s: not significant, *: p<0.05. (MeaniSD, n=3 invasion assay devices/condition/cell line in one experiment; two independent experiments); (C) GKAP mRNA was knocked down in mPDAC-4361 (i), and in two hPDAC cell lines, DanG (ii) and SUIT2 (iii). The knockdown efficiency was assessed by Western blot analysis; numbers below indicate levels of GKAP normalized to GAPDH. Cell invasiveness in the invasion assays was shown in bar graphs (mPDAC line: mean±SD. Two-way ANOVA, Bonferroni’s multiple comparisons test, *: p<0.05, ****: p<0.0001 ; hPDAC lines: mean ± SEM, unpaired t test, *: p<0.05, **: p<0.01. For all invasion assays: n=3 invasion assay devices/condition in one experiment. Two independent experiments.) Representative images of DAPI stained nuclei from the invasion assay illustrate the cells that reached the other side of the membrane of a Boyden chamber (i, right panels);
(D) Treatment with MK801 decreased HSF1 phosphorylation as well as FMRP expression. The numbers below indicate levels of pHSF1 and FMRP normalized to GAPDH (n= 3);
(E) Western blot analysis assessing siRNA-mediated HSF1 and FRMP knockdown (HSF1 (human) siRNA, Thermo Fisher Scientific, #4392420, Assay ID s6951 , s6952 ; FMRP/ fmr1 (human) siRNA, Thermo Fisher Scientific, #4392420, Assay ID s5316), in DanG cells (i) and SUIT2 cells (ii) are shown in the left panels. The numbers below indicate levels of HSF1 and FMRP normalized to GAPDH. The knockdowns decreased cancer cell invasion in the flow-guided invasion assay (bar graphs on the right panels). (MeaniSEM, unpaired t test, *: p<0.05, **: p<0.01. n=3 invasion assay devices/condition in one experiment. Two independent experiments were performed with consistent results);
Figure 6 shows that the NMDAR/GKAP/FMRP/HSF1 signaling axis is active in PDAC tumours:
(A) GKAP, FMRP and HSF1 are highly expressed in mPDAC GEMM, both in primary PDAC tumours (upper panels) and in liver metastases (lower panels). The primary tumour panels are representative of >5 tumour fields/pancreas from >20 mice. The liver metastasis panels are representative of two liver macro-metastases (~1cm in diameter);
(B) p-GluN2b, GKAP, FMRP and HSF1 were all detected in hPDAC tumours displayed in a tissue microarray (i). The images were quantified to show the percentage of positive areas on each tissue section, and the expression of all four proteins was further elevated in the progression from primary to lymph node metastatic PDAC in humans (ii). The expression of GKAP, FMRP and HSF1 all positively correlated with p-GluN2b expression (iii). p-GluN2b was also associated with larger tumour size (iii) and with vascular invasion by cancer cells (iv), classified as absent (VO) or present (V1);
Figure 7 shows the identification of gene expression signatures for mPanNETs: (A) Schematic presentation for gene expression signatures associated with tumour phenotypes. Bold italic and normal boxes mark the samples used in the signature analysis; bold italic suggests enrichment of the gene expression signature, while normal suggests depletion;
(B) Mouse Strain Signature: (i) Heat map showing major drivers of the strain signature (fold change >2; |Z| >9). (ii) Box-plot demonstrating the sample groups and the distribution of their corresponding Z-scores. (iii) The top driver genes (|Z|>9) in the signature are shown. Dlgap1/GKAP clearly associates with the strain signature {bold italic, up-regulated in B6; normal: up-regulated in C3H);
(C) MK801 Treatment Signature: (i) Heat map showing the 330“MK801 treatment signature” genes, which segregated MK801 -treated B6 tumour samples from B6 control tumours, (ii) Box-plot demonstrating the sample groups and the distribution of their corresponding Z-scores. (iii) The top driver genes (|Z|>9) in the signature are shown {bold italic, up-regulated in MK801-treated B6 tumours; normal: up- regulated in B6 control tumours);
(D) Leading edge analysis identified 148 common driver genes in both“MK801 treatment signature” and “mouse strain signature”, representing “NMDAR- pathwayl0W signature” (Table S4). Shown here are the“core” common driver genes, which have |Z|>3 in both signatures {bold italic, up-regulated in MK801 -treated B6 tumours and C3H tumours; normal: up-regulated in B6 controls);
Figure 8 shows that the signatures of low-/inhibited- NMDAR activity is associated with favorable prognosis in human cancer types:
(A) In a mouse model of PDAC, both MK801 (left panel) and memantine (right panel) treatments prolonged the survival of PDAC-bearing mice. (Left: control group: 28 mice, median survival: 23 days after enrollment; MK801 group: 25 mice, median survival: 36 days after enrollment. p=0.0206, Log-rank test. Right: control group: 35 mice, median survival 13.4 weeks; memantine group: 33 mice, median survival 15.4 weeks. Log-rank test, p=0.0469);
(B) PDAC patients whose tumours correlated with the mPanNET“MK801 treatment signature” exhibited significantly better survival compared to the remaining patients (n=13 for associated, n=165 for not associated);
(C) COX regression analysis demonstrated that the“MK801 treatment signature” was significantly associated with patient prognosis, both in univariate and multivariable analyses;
(D) Expression of the“MK801 treatment signature” was associated with favorable prognosis in patients from several cancer types in addition to PDAC, including glioma (combining low grade glioma and glioblastoma) and kidney cancers (combining three major subtypes of kidney cancer: chromophobe renal cell carcinoma, clear cell renal carcinoma and papillary kidney carcinoma). All patients were included in each cancer type shown, regardless of treatment and staging (Brain cancer, associated: n=575, not associated: n=89; kidney cancers, associated: n=504, not associated: n=381 );
(E) Cumulative distribution function (CDF) plot demonstrating that low-grade gliomas (full line) are significantly more associated with the (pathway-low) MK801 - treatment signature compared to high grade glioblastomas (dotted line) (p < 2.22e- 16);
(F) PDAC patients whose tumours correlated with the mPanNET “NMDAR- pathwaylow signature” (Figure 7E) similarly exhibited significantly better survival times compared to the remaining patients, as in the larger signature utilized in 7B (n=13 for associated, n=164 for not associated);
(G) The “NMDAR-pathwayl0W signature” was also associated with favorable prognosis in patients from several cancer types. All patients were included in each cancer type shown, regardless of treatment and staging (Brain cancer, associated: n=572, not associated: n=88; kidney cancers, associated: n=501 , not associated: n=378; UVM: uveal melanoma, associated: n= 71 , not associated: n=9);
Figure 9 shows that GKAP/dlgap1 is the most differentially expressed invasion modifier gene:
(A) Re-evaluation of the qRT-PCR data from Chun et al. revealed that GKAP (Dlgapl ) is the most differentially expressed genetic modifier on Chromosome 17. The Y-axis indicates the log of the relative ratio of expression in B6 compared to C3H tumours and normal pancreatic islets (B6/C3H). Therefore, a positive value indicates high expression in the B6 background, whereas a negative value indicates the opposite. Arrowhead denotes GKAP (Dlgapl ) expression levels;
(B) qRT-PCR comparative analysis of GKAP mRNA in organs from wild-type
C57BI/6 (B6) and C3Heb/Fe (C3H) mouse strains. Two-way AN OVA with Bonferroni multiple comparisons test. Mean with SEM. ***: p<0.001 , ****: p<0.0001 . (n=4-7 mice per analysis). Note that in the case of the pancreas RNA was extracted from whole tissue, which is predominantly composed of pancreatic acinar cells;
Figure 10 shows that FMRP and HSF1 are downstream effectors of the
NMDAR/GKAP pathway: (A) qRT-PCR demonstrates efficient GKAP knockdown in * TC-3 cells. (n=3 technical controls for qRT-PCR; 2 independent knockdowns were generated, with similar trend. ****: p<0.0001 . Unpaired t-test. Bars shown are means with SEM);
(B) Western blot analysis for FMRP in B6 compared to C3H PanNETs. Mann- Whitney test, *: p<0.05 (Each lane shows one tumour pool from one mouse, consisting of more than 3 tumours/pool, and 6 mice/group);
(C) IHC staining of FMRP in B6 PanNETs. (i) Overview of the whole pancreas, (ii) From the same tumour (the IC1 tumour in the center), close-up of FMRP staining at the invasive front (left panels) and at the non-invasive border (right panels). LN: lymph node; IT: islet tumour; IC1 : invasive carcinoma type 1 (focally invasive); IC2: invasive carcinoma type 2 (highly invasive);
(D) IHC staining of FMRP in a hyperinvasive B6 PanNET observed after sunitinib treatment;
(E) Microarray data from Sadanandam et al. (Sadanandam et al., 2015) demonstrated that expression of Fmr1 (the gene encoding FMRP) was gradually elevated during the multistage tumourigenesis pathway that leads to PanNETs in B6 mice;
(F) IHC quantification of FMRP staining was performed in 10 images of non-invasive tumours, 23 images of invasive tumours and 7 images from metastatic lesion. (*: p<0.05, ****: p<0.0001 . 1 -way ANOVA, Kruskal-Wallis test, compared to non- invasive tumours);
(G) qRT-PCR for Fmr1/FMRP and Dlgap1/GKAP transcripts after shRNA knockdown of GKAP in · TC-3 (Openbiosystem, Clone ID: TRCN0000088935.). (For each gene, the expression levels in the siRNA-HSF1 (Thermo Fisher Scientific, #4390771 , Assay ID s67870, s67871 ) groups were normalized to siRNA-control
(Thermo Fisher Scientific, #AM461 1 , AM4613). One column statistics, comparing with a hypothetical value of 1 . **: p<0.01 ; n.s.: not significant. n=3 independent RNA extraction/condition); Figure 11 shows distinct gene expression signatures corresponding to different tumour phenotypes:
(A) Gene ontology analysis using GeneGo Metacore software (Thomson Reuters, https://portal.genego.com/) predicted that genes within the“NMDAR-pathwaylow signature” were associated with“Neurogenesis/Synpatogenesis”;
(B) Isoform analysis showed that all three GKAP/Dlgap1 protein-coding isoforms expressed in mPanNETs were significantly differentially expressed (FDR=0) between tumours from B6 and C3H strains, with B6 PanNETs having higher expression compared to C3H PanNETs;
Figure 12 shows that MK801 has therapeutic benefit in PDAC GEMM; MK801 treatment signature was associated with better survival in patients:
(A) CDF plot demonstrating that the “MK801 treatment signature” was more associated with low grade (T1/T2) tumours (dotted line) than with high grade (T3/T4) tumours (full line) in pancreatic ductal adenocarcinoma patients (Using genes with |Z|>4, and log2 fold change (MK801/control) > 1);
(B) Kaplan-Meyer plots showing overall survival in various cancer types from the TCGA (http://cancergenome.nih.gov/). Gene expression data from patient cohorts was stratified by their enrichment for the“MK801 treatment signature” identified in the mPanNET RNA-seq analysis;
Full line: patients whose tumours had gene expression most correlated with the MK801 treatment signature (defined by the top 3.5 Z score). Dotted line: patients whose tumours had gene expression least correlated with the MK801 treatment signature (defined by the bottom 3.5 Z score). All patients were included in each cancer type shown, regardless of treatment and staging;
(C) Kaplan-Meyer plots showing overall survival in various cancer types from the
TCGA (http://cancergenome.nih.gov/). Gene expression data from patient cohorts was stratified by their enrichment for the“NMDAR-pathwayl0W signature” identified in the mPanNET RNA-seq analysis. Full line: patients whose tumours had gene expression most correlated with the core driver gene signature (defined by the top 3.5 Z score); dotted line: patients whose tumours had gene expression least correlated with the core driver gene signature (defined by the bottom 3.5 Z score). All patients were included in each cancer type shown, regardless of treatment and staging. LLC: low grade glioma.
Detailed description of the Invention
The present disclosure may be more readily understood by reference to the following detailed description presented in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular aspects by way of example only and is not intended to be limiting of the claimed disclosure.
As used herein and in the appended claims, the singular forms "a", "an" and
"the" include plural referents unless the context clearly dictates otherwise. Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise", "comprises", "comprising", "include", "includes" and "including" are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various aspects use the term "comprising", those skilled in the art would understand that in some specific instances, an aspect can be alternatively described using language "consisting essentially of or "consisting of."
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, unless otherwise required by the context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.
A "gene" is defined as a nucleic acid molecule that comprises a nucleic acid sequence that encodes an RNA molecule and the expression control sequences that surround the nucleic acid sequence that encodes the RNA molecule. The encoded RNA molecule may be a functional RNA (e.g. tRNA, rRNA), regulatory RNA (e.g. siRNA, miRNA, tncRNA, smRNA, snRNA) or messenger RNA (mRNA). Messenger RNA molecules may be transcribed into polypeptides which are also considered as being encoded for by the gene. For instance, a gene may comprise a promoter, one or more enhancers, a nucleic acid sequence that encodes an RNA molecule, downstream regulatory sequences and, possibly, other nucleic acid sequences involved in regulation of the expression of an RNA. As is well known in the art, eukaryotic genes usually contain both exons and introns. The term "exon" refers to a nucleic acid sequence found in genomic DNA that is bioinformatically predicted and/or experimentally confirmed to contribute contiguous sequence to a mature RNA transcript. The term "intron" refers to a nucleic acid sequence found in genomic DNA that is predicted and/or confirmed to not contribute to a mature RNA transcript, but rather to be "spliced out" during processing of the transcript.
A "gene product" is defined as a molecule expressed or encoded directly or indirectly by a gene. For example, gene products include pre-mRNA, mature mRNA, tRNA, rRNA, snRNA, uIRNA, siRNA, miRNA, tncRNA, smRNA, prepolypeptides, pro-polypeptides, mature polypeptides, post translationally modified polypeptides, processed polypeptides, functionally active polypeptides, functionally inactive polypeptides, and complexed polypeptides. A single gene product may have several molecular functions and different gene products may share a single or similar molecular function. Preferably, the gene product also encompasses variants and/or fragments thereof.
As used herein, the term“variant” refers to biologically active derivatives of a nucleic acid sequence or polypeptide sequence of the invention. In general, the term“variant” refers to molecules having a native sequence and structure with one or more additions, substitutions (generally conservative in nature) and/or deletions (e.g. splice variants), relative to the native molecule, so long as the modifications do not destroy biological activity and which are“substantially homologous” to the reference molecule. In general, the sequences of such variants are functionally, i.e. biologically, active variants and will have a high degree of sequence homology to the reference sequence, e.g., sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90% or 95% or more, when the two sequences are aligned.
Alternatively, the term“variant” also refers to post-transcriptionally modified nucleic acid sequence (e.g. methylation, phosphorylation, etc..) or polypeptide sequence (e.g. isoform, ...) of the invention.
As used herein, a“fragment” of one or more nucleic acid sequence or polypeptide sequence of the invention refers to a sequence containing less nucleotides or amino acids in length than the respective sequences of the invention while retaining the biological activity described herein. Preferably, this fragment contains, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid sequence or polypeptide sequence.
As used herein, the wording “differential transcription and/or expression and/or activity of a gene or gene product” and their synonyms, which are used interchangeably, refer to one or more gene(s) or gene product(s) whose transcription and/or expression and/or activity is/are activated to a higher or lower level in a subject suffering from a disease, specifically cancer, relative to its/their transcription and/or expression and/or activity in a normal or control subject, or reference data. The terms also include genes or gene products whose transcription and/or expression and/or activity is altered to a higher or lower level at different stages of the same disease. It is also understood that a differentially transcripted and/or expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alternative splicing to result in a different polypeptide product. Such differences may be evidenced by a change in mRNA levels, surface expression, secretion or other partitioning of a polypeptide, for example.
Differential gene transcription and/or expression and/or activity may include a comparison of transcription and/or expression and/or activity between two or more genes or their gene products, or a comparison of the ratios of the transcription and/or expression and/or activity between two or more genes or their gene products, or even a comparison of two differently processed products of the same gene, which differ between normal subjects and subjects suffering from a disease, specifically cancer, or between various stages of the same disease. In particular, the reference (“control”) transcription and/or expression and/or activity level can be the transcription and/or expression and/or activity level of the gene product in a control sample. The control sample can be a normal sample, that is, a non-tumoural sample, preferably from the same tissue than the cancer sample, or a basal level of transcription and/or expression and/or activity.
The normal sample may be obtained from the subject affected with the cancer or from another subject, such as a normal or healthy subject, i.e. a subject who does not suffer from a cancer. Additionally or alternatively, the control sample can be obtained from data repositories of cancer patient expression studies; accordingly, the control sample could be in this case a virtual sample obtained from such a data repository for a given cancer type. Transcription and/or expression and/or activity levels obtained from cancer and normal samples may be normalized by using e.g. expression levels of proteins which are known to have stable expression such as RPLPO (acidic ribosomal phosphoprotein PO), TBP (TATA box binding protein), GAPDH (glyceraldehyde 3- phosphate dehydrogenase) or b-actin. Additionally or alternatively, as it will be appreciated by a person skilled in the art, it is possible to use statistical methods based on whole-transcriptome expression distribution for normalization of cancer and control/normal samples.
Differential transcription and/or expression and/or activity includes both quantitative, as well as qualitative, differences in the temporal or cellular transcription and/or expression and/or activity pattern in a gene or its products among, for example, normal and diseased cells, or among cells which have undergone different disease events or disease stages. For the purpose of this description, “differential gene transcription and/or expression and/or activity” is considered to be present when there is at least a two-fold difference between the transcription and/or expression and/or activity of a given gene or gene product in normal and diseased subjects, or in various stages of disease development in a diseased subject. As a way of example, in some tumours the normal tissue/cell-of-origin may not express the NMDAR pathway; rather, the pathway may be upregulated and activated during the cancerous transformation process, but importantly to different degrees, producing signature-LOW vs signature-HIGH tumours with differing prognoses. Accordingly, NMDAR-pathway-LOW signatures can be defined in comparison to other tumours of the same type that are NMDAR-pathway-HIGH.
The term“over-expression” with regard to an RNA transcript is used to refer to the level of the transcript determined by normalization to the level of reference mRNAs, which might be all measured transcripts in the specimen or a particular reference set of mRNAs. The term "percent sequence identity" in the context of nucleic acid or amino acid sequences refers to the residues in two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine, usually at least about 20, more usually at least about 24, typically at least about 28, more typically at least about 32, and preferably at least about 36 or more nucleotides or amino acids. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. In the frame of the present disclosure, the terms "percent sequence identity", "percent sequence similarity" and "percent sequence homology" can be used interchangeably.
As used herein, the terms "patient" or“subject” refer to an animal, preferably to a mammal, even more preferably to a human, including adult and child. However, the terms can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheeps and non-human primates, among others, that are in need of treatment.
The term "sample", as used herein, means any sample containing cells, nucleic acids and/or proteins derived from a subject. Examples of such samples include fluids such as blood, plasma, saliva, and urine as well as biopsies, organs, tissues or cell samples.
The term“tumour” or“cancer”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Additionally or alternatively, the terms refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. The term “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, blood cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, and brain cancer.
As used herein, a cancer is said to be “aggressive” if it forms, grows, or spreads quickly. Aggressive cancers are characterized by a high malignancy, i.e. marked metastasizing and invasive properties correlated with a poor prognosis. As used herein, a cancer is said to be“invasive” if it has the ability to disrupt and spread beyond a basement membrane, or beyond its confines into adjacent normal tissue. Invasive cancers generally carry a poorer prognosis than non-invasive cancers, since invasive cancers are not delimited by basement membrane barriers and can metastasize to other areas of the body. Being able to predict whether a cancer is aggressive allows the oncologist to accurately formulate an appropriate treatment regimen based on the cancer’s likelihood of spreading and having a poor prognosis. Thus, an invasive cancer would generally be treated more aggressively than a cancer that will not spread.
The term "prognosis" defines a forecast as to the probable outcome of a disease, the prospect as to recovery from a disease, or the potential recurrence of a disease as indicated by the nature and symptoms of the case. The term further refers to the prediction of the likelihood of cancer-attributable death or progression, including recurrence, metastatic spread and/or drug resistance, of a neoplastic disease. The term“prediction” is used herein to refer to e.g. the likelihood that a patient will respond either favourably or unfavourably to a drug or set of drugs, and also the extent of those responses, or that a patient will survive, following surgical removal or the primary tumour and / or chemotherapy for a certain period of time without cancer recurrence. The predictive described herein can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient.
As used herein, the term "good prognosis" refers to a probable favourable outcome of a disease, recovery from a disease or low potential for disease recurrence such as an increased patient survival and/or a late disease progression and/or a and/or a decreased metastasis formation. The term "poor prognosis" indicates refers to a non-favourable outcome of a disease or non-recovery from a disease, such as a decreased patient survival and/or an early disease progression and/or an increase disease recurrence and/or an increase metastasis formation.
As used herein, "treatment”, "treating” and the like generally refers to any act intended to ameliorate the health status of subjects such as an animal, particularly a mammal, more particularly a human, such as therapy, prevention, prophylaxis and retardation of the disease, and includes: (a) inhibiting the disease, i.e., arresting its development; or (b) relieving the disease, i.e., causing regression of the disease and/or its symptoms or conditions such as improvement or remediation of damage. In certain aspects, such term refers to the amelioration or eradication of a disease or symptoms associated with a disease. In other aspects, this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease. The term “prevention” or“preventing” relates to hampering, blocking or avoid a disease from occurring in a subject which may be, for any reason, predisposed to the disease but has not yet been diagnosed as having it for example based on familial history, health status or age. In order to address the limitations concerning the understanding of the involvement of genetic factors in cancer invasiveness and progression, the inventors established a research program aimed at identifying genetic signatures and the influence of the genetic background capable to explain the PanNET mouse model phenotype, as well as their contribution in both animal models and human counterparts in tumour malignancy, namely the capability for tissue invasion (and metastasis). The subject-matter described in the present disclosure is based, at least in part, on the surprising evidence that, in mouse models, GKAP is a potential modifier gene of NMDAR pathway activity whose differential expression contributes to genetic background-specific differences in pancreatic tumour invasiveness. Combining pharmacological inhibition of NMDAR and genetic knockdown of GKAP in cancer cells, the inventors elucidated the autocrine glutamate-NMDAR-GKAP signalling axis and its functional role in the invasive/malignant phenotypes of both mouse and human pancreatic ductal adenocarcinoma (PDAC). Finally, the inventors have identified a gene expression signature for inhibited/low NMDAR signalling in the PanNET transcriptome, which is significantly associated with favourable patient survival in several human cancers, including PDAC. This association is useful for predicting the aggressiveness of a cancer and assessing treatment options. The present disclosure therefore provides a set of genes and gene products, the expression, transcription or blocking/inhibition of which is of prognostic value.
In one aspect, the subject-matter described in this specification provides for a method for determining the prognosis for survival of a patient having a cancer, said cancer being altered (i.e. highly activated or alternatively inactive/inhibited) for what concerns the NMDAR signalling pathway, said method comprising a step of determining the transcription level and/or expression level and/or the activity of a plurality of genes or gene products selected from genes listed in Tables 1 to 4 in a sample from said cancer patient relative to a control, wherein the differential transcription and/or expression and/or activity of said plurality of genes or gene products relative to a control is indicative of cancer aggressiveness and therefore of the patient’s prognosis. The differential transcription and/or expression and/or activity of said plurality of genes or gene products relative to a control can be up- regulation or down-regulation of said transcription and/or expression and/or activity of said genes or gene products compared to a control level such as for instance a basal level.
The "NMDAR signalling pathway", as referred to herein, refers to a series of biochemical events, as well as to the signalling biochemical elements involved in said events, downstream to the NMDA receptor (“NMDAR”) activation. This signalling pathway is intended to include molecular events from activation of the NMDA receptor to end effects such as e.g. cell proliferation and/or invasion, or intermediate effects such as Ca2+ cell influx or phosphorylation of NMDA receptor subunits.
As referred to herein the phrase "altered in the NMDAR signalling pathway" is intended to mean the alteration of one or more signalling elements in the pathway (e.g. to affect its enzymatic, structural or other functional properties) which affects downstream signalling events. Alteration of the signalling elements refers to e.g. the ability to form interactions with other molecules, e.g. protein-protein interactions. The ultimate effect is an alteration of downstream events which typify the NMDAR signalling. Alteration of said signalling pathway may be assessed for example by determining the extent of activation of a molecule involved in said pathway, examination of levels of molecules whose levels are dependent on the activity of said pathway and the like.
An altered NMDAR signalling pathway can be a highly activated NMDAR signalling pathway or alternatively an inactive/inhibited or less active NMDAR signalling pathway compared to any suitable control such as a baseline, normal level. As a way of example, a highly activated NMDAR signalling pathway is present in concomitance with a high GKAP expression in tumour cells, which leads to deregulated protein translation and elevated FMRP/p-HSF1 expression, and finally heightens a tumour cell invasive behaviour. On the contrary, an inactive/inhibited or less active NMDAR signalling pathway is present in tumour cells expressing low GKAP, having low NMDAR pathway activity, and which are mostly non-invasive in nature.
One of the surprising evidences upon which the present disclosure is based, at least in part, is the fact that GKAP acts as an effector of NMDAR signalling that is instrumental for the invasive tumour growth in both neuroendocrine and pancreatic ductal cancers, as well as the molecular mechanisms involved in these cellular responses. It has been established by the present inventors that GKAP governs invasive growth and treatment response to NMDAR inhibitors of PanNET via its pivotal role in regulating NMDAR pathway activity. It has further been acknowledged that downstream effectors FMRP and HSF-1 function along with GKAP to support invasiveness of PanNET and pancreatic ductal adenocarcinoma cancer cells.
Furthermore, it has been assessed by the inventors that gene signatures exist that correlates with an invasive or non-invasive phenotype, being indicative of active vs. inactive/inhibited/less active NMDAR signalling, of pancreatic cancer, and in turn to a patient’s prognosis. Via genome-wide expression profiles orchestrated by the NMDAR-GKAP signalling axis, said transcriptome signatures were identified in tumours with low/inhibited NMDAR activity that significantly associate with favourable patient prognosis in several cancer types such as brain cancers (e.g. glial brain cancer), kidney cancers, skin cancer like uveal melanoma and blood cancer like acute myeloid leukaemia (AML). Flowever, the present disclosure is not intended to be limited to the reported examples, and it is understood that the identified signatures can be used to determine cancer invasiveness as well as patient’s prognosis for other type of cancers such as breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract and/or thyroid cancer. Those gene signatures have been identified in a mouse model of pancreatic cancer as four sets of gene products altered in their expression due to the genetic background of the tested subject and/or due to a treatment of the tested subject with a prototypical antagonist of the NMDA receptor, MK-801 (also known as Dizocilpine). As a first step, a so-called “strain signature” was identified, distinguishing gene products differently modulated in tumour samples from mouse strains having different genetic backgrounds (mouse strains B6 and C3H). It was later assessed, on the mouse model strain having the genetic background providing the most invasive tumour progression (B6 mouse strain), that some gene products are modulated (i.e. up-/down-regulated or up-/down-expressed) depending on the treatment with MK-801 , defining a so-called“MK-801 treatment signature”. Within said signature, a sub-signature has been identified, defining a so-called“NMDAR- pathwayl0W gene signature”, in which gene products are oppositely expressed in B6 mice compared to C3H mice (having the genetic background providing the less invasive tumour progression), as well as compared to B6 mice treated with MK-801 . Said gene signatures identified in mouse models have been later on validated on cancer samples from humans, thus confirming the results of the initial findings also in higher mammals.
The gene signatures according to the present disclosure have been grouped herein as follows:
TABLE 1 : list of genes whose gene products are up-regulated in MK801 treatment signature;
TABLE 2: list of genes whose gene products are down-regulated in MK801 treatment signature;
TABLE 3: list of genes whose gene products are up-regulated in NMDAR-pathwaylow gene signature;
TABLE 4: list of genes whose gene products are down-regulated in NMDAR- pathwayl0W gene signature. Accordingly, in one aspect, the down-regulation of a plurality of genes or gene products selected from the genes listed in Table 1 and/or 3, and/or up-regulation of a plurality of genes or gene products selected from the genes listed in Table 2 and/or 4, correlates with a high activation of the NMDAR signalling pathway in the cancer cells and therefore with a poor prognosis, whereas up-regulation of a plurality of genes or gene products selected from the genes listed in Table 1 and/or 3, and/or down-regulation of a plurality of genes or gene products selected from the genes listed in Table 2 and/or 4 correlates with a low activation of the NMDAR signalling pathway in the cancer cells and therefore with a favourable prognosis.
The predictive methods of the present disclosure are valuable tools in predicting if a subject like a human cancer patient is likely to respond favourably to a treatment regimen, such as surgical intervention, chemotherapy with a given drug or drug combination, and/or radiation therapy, or whether long-term survival of the patient, following surgery and/or termination of chemotherapy or other treatment modalities, is likely.
In particular, the predictive methods of the present disclosure are useful for selecting a subject affected with a cancer for a therapy and/or in determining whether a subject like a human patient is likely to respond favourably to, or benefit from, a treatment regimen based on at least one of a plurality of active compounds, such as agonists or antagonists of:
- at least one of a plurality of genes or gene products listed in Tables 1 to 4; and/or
- a gene or gene product member of the NMDA receptor signalling pathway; and/or
- a gene or gene product member of the DLGAP family of genes; and/or
- a HSF1 gene or gene product; and/or
- a FMR1 gene or gene product.
As used herein, the term "GKAP/DLGAP1" refers to guanylate kinase-associated protein, also known as Disks large-associated protein 1 (DAP-1). Accession numbers corresponding to the human GKAP/DLGAP1 gene in Genbank are NM_004746.3, NM_001003809.2, NM_001242761.1 , NM_001242762.1 ,
NM_001242763.1 , NM_001242764.1 , NM_001242765.1 , NM_001242766.1 ,
NM_001308390.1 (in total 9 transcript variants), and accession numbers corresponding to the human GKAP proteins are NP_004737, NP_001295319, NP_001229692, NP_001003809, NP_001229693, NP_001229690,
NP_001229695, NP_001229691 , NP_001229694. This proteins are encoded by the gene DLGAP1 (GenelD: 9229). GKAP is a protein known to be highly enriched in synaptosomal preparations of the brain, and it has been shown to facilitate the assembly of the post synaptic density of neurons. The term“DLGAP family genes” refers to DLGAP1 and other four structurally-related genes, including DLGAP2 (GenelD: 9228), DLGAP3 (GenelD: 58512), DLGAP4 (GenelD: 22839) and DLGAP5 (GenelD: 9787).
As used herein, the term "HSF1" refers to the heat shock transcription factor 1. Accession number corresponding to the human HSF1 gene in Genbank is NM_005526, and accession number corresponding to the human HSF1 protein is
NP_005517. This protein is encoded by the gene HSF1 (GenelD: 3297). The product of this gene is a transcription factor that is rapidly induced after temperature stress and binds heat shock promoter elements. HSF1 is the primary mediator of transcriptional responses to proteotoxic stress with important roles in non-stress regulation such as development and metabolism. The HSF1 protein regulates the heat shock response (HSR) pathway in humans by acting as the major transcription factor for heat shock proteins, ensuring proper folding and distribution of proteins within cells. This pathway is induced by not only temperature stress, but also by a variety of other stressors such as hypoxic conditions and exposure to contaminants. HSF1 transactivates genes for many cytoprotective proteins involved in heat shock, DNA damage repair, and metabolism. This illustrates the versatile role of HSF1 in not only the heat shock response, but also in aging and diseases such as cancer. As used herein, the term "FMRP" refers to the fragile X mental retardation protein. Accession numbers corresponding to the human FMRP gene (FMR1 ) in Genbank are NM_001 185076, NMJD02024, NM_001 185082, NM_001 185075,
NM_001 185081 , and accession numbers corresponding to the human FMRP proteins are NP_001 172005, NP_002015, NP_001 17201 1 , NP_001 172004, NP_001 172010. This protein is encoded by the gene FMR1 (GenelD: 2332). FMRP, most commonly found in the brain, is essential for normal cognitive development and female reproductive function. FMRP has been suggested to play roles in nucleocytoplasmic shuttling of mRNA, dendritic mRNA localization, and synaptic protein synthesis. The proposed mechanism of FMRP’s effect upon synaptic plasticity is through its role as a negative regulator of translation; FMRP is an RNA- binding protein which associates with polyribosomes and has been shown to inhibit translation of mRNA. Mutations of this gene can lead to fragile X syndrome, intellectual disability, premature ovarian failure, autism, Parkinson's disease, developmental delays and other cognitive deficits.
In one aspect, the gene product is a polypeptide. The quantity of protein may be measured by semi-quantitative Western blots, enzyme-labelled and mediated immunoassays, such as ELISAs, biotin/avidin type assays, radioimmunoassay, Immunoelectrophoresis or immunoprecipitation or by protein or antibody arrays. The protein expression level may be assessed by immunohistochemistry on a tissue section of the sample (e.g. frozen or formalin-fixed paraffin embedded material). The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. Preferably, the quantity of protein is measured by immunohistochemistry or semi-quantitative western-blot.
In another aspect, the gene product is mRNA. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the sample (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR). In some aspects, quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous. Preferably, primer pairs were designed in order to overlap an intron, so as to distinguish cDNA amplification from putative genomic contamination. Other primers may be easily designed by the skilled person. Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). Preferably, the quantity of mRNA is measured by quantitative or semi-quantitative RT-PCR or by real-time quantitative or semi-quantitative RT-PCR or by transcriptome approaches. Besides real-time quantitative or semi-quantitative RT-PCR, RNA-sequencing (RNAS_seq) is commonly used as well, and it is herein preferred.
In various aspects, the expression level and/or the transcription level and/or the activity of a plurality of genes or gene products is determined, said plurality of genes or gene products comprising at least 2, or at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30, or at least 35, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100, or at least 150, or at least 200, or at least 250, or at least 300 prognostic genes or gene products. In one aspect, said plurality of genes or gene products comprises or consists of the totality of the 330 genes or gene products listed in Tables 1 and 2. In an alternative or additional aspect, said plurality of genes or gene products comprises or consists of the totality of the 148 genes or gene products listed in Tables 3 and 4. In some aspects, said plurality of genes or gene products comprises or consists of the following genes or gene products:
a) Fam107a, Tex14, Trpc5, Zbtb16, Zcchc16, Adm2, Bcatl and Hhip; and/or b) Dsp, Eif2s3y, Fam107a, Hunk, Olig3, Rbfoxl and Zbtb16; and/or
c) Ifi202b, H2-Ea-ps, Klk1 b22, Gm8615, H28, H2-BI, Pyy, Gm8801 , Hist1 h2bk and LOC547349; and/or
d) Adamded , Dlgapl , Trim12a, 31 10007F17Rik, Rhox4a, Ccl21 b, Folrl , Strc and S100z; and/or
e) Rbm44, Gpr81/Hcar1 , Rec8, Sycel , Ccl8, Cled Oa, Retnla, Ccl7, Clec3b, Ccr2 and Cd209f; and/or
f) Gpx2, Klra17, Lin28b, Olfm3, Prssl , Rbm44, Rec8, Slitrk2 and Sycel (see Figure 7).
In particular, the modulation of the expression level and/or the transcription level and/or the activity of genes or gene products of the above comprises increasing the expression level and/or the transcription level and/or the activity of genes or gene products whose up-regulation or over-expression is associated with a good prognosis, and in a preferred aspect the genes or gene products are selected from a group comprising or consisting of the genes or gene products listed in Tables 1 and/or 3.
In one aspect, the genes or gene products whose up-regulation or over- expression is associated with a good prognosis, and for which increase in the expression level and/or the transcription level and/or the activity is sought, comprise or consist of the following:
a) Fam107a, Tex14, Trpc5, Zbtb16, Zcchc16, Adm2, Bcatl and Hhip; and/or b) Dsp, Eif2s3y, Fam107a, Hunk, Olig3, Rbfoxl and Zbtb16; and/or
c) Ifi202b, H2-Ea-ps, Klk1 b22, Gm8615, H28, H2-BI, Pyy, Gm8801 , Hist1 h2bk and LOC547349. Additionally or alternatively, the modulation of the expression level and/or the transcription level and/or the activity of genes or gene products comprises decreasing the expression level and/or the transcription level and/or the activity of genes or gene products whose down-regulation or down-expression is associated with a good prognosis, and in a preferred aspect the genes or gene products are selected from a group comprising or consisting of the genes or gene products listed in Tables 2 and/or 4.
In one aspect, the genes or gene products whose down-regulation or down- expression is associated with a good prognosis, and for which decrease in the expression level and/or the transcription level and/or the activity is sought, comprise or consist of the following:
d) Adamded , Dlgapl , Trim12a, 31 10007F17Rik, Rhox4a, Ccl21 b, Folrl , Strc and S100z; and/or
e) Rbm44, Gpr81/Hcar1 , Rec8, Sycel , Ccl8, Cled Oa, Retnla, Ccl7, Clec3b, Ccr2 and Cd209f; and/or
f) Gpx2, Klra17, Lin28b, Olfm3, Prssl , Rbm44, Rec8, Slitrk2 and Sycel .
Accordingly, the subject-matter described in the present specification further provides a method for improving the prognosis for a cancer patient being diagnosed to have a poor prognosis, comprising modulating the expression level and/or the transcription level and/or the activity of:
- a plurality of genes or gene products, or at least one of said genes or gene products, listed in Tables 1 to 4; and/or
- a gene or gene product member of the NMDA receptor signalling pathway; and/or
- a gene or gene product member of the DLGAP family of genes; and/or
- a HSF1 gene or gene product; and/or
- a FMR1 gene or gene product.
Preferably, said modulation is carried out by administering to a cancer patient: a. an inhibitor of at least one gene, or gene product derived therefrom, listed in Tables 2 and/or 4; and/or
b. at least one gene, or gene product derived therefrom, listed in Tables 1 and/or 3, or an activator thereof; and/or
c. an inhibitor of the NMDA receptor; and/or
d. an inhibitor of a gene product member of the NMDA receptor signalling pathway; and/or
e. an inhibitor of a member of the DLGAP family of genes or of gene product(s) derived therefrom; and/or
f. an inhibitor of the HSF1 gene or of gene product(s) derived therefrom; and/or g. an inhibitor of the FMR1 gene or of gene product(s) derived therefrom.
The method can be used to improve the prognosis of a patient being affected by a cancer selected from a list comprising blood cancer such as acute myeloid leukaemia, breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma such as uveal melanoma, and brain cancer such as glial brain cancer.
Accordingly, one aspect of the subject-matter described in the present specification relates to a pharmaceutical composition for use in the treatment of a cancer in a patient being diagnosed to have a poor prognosis, comprising:
a. an inhibitor of at least one gene, or gene product derived therefrom, listed in Tables 2 and/or 4; and/or
b. at least one gene, or gene product derived therefrom, listed in Tables 1 and/or 3, or an activator thereof; and/or
c. an inhibitor of the NMDA receptor; and/or d. an inhibitor of a gene product member of the NMDA receptor signalling pathway; and/or
e. an inhibitor of a member of the DLGAP family of genes, or of gene product(s) derived therefrom; and/or
f. an inhibitor of the HSF1 gene, or of gene product(s) derived therefrom; and/or
g. an inhibitor of the FMR1 gene, or of gene product(s) derived therefrom. Said inhibitors or activators can be selected from a non-exhaustive list comprising a nucleic acid molecule, a polypeptide, a fusion protein, an antibody as well as a derivative or fragment(s) thereof.
In one aspect, said inhibitor is selected from a group including but not limited to MK801 , memantine, Ifenprodil, ketamine and Ro 25-6981.
The pharmaceutical composition can be used to improve the prognosis of a patient being affected by a cancer selected from a list comprising blood cancer such as acute myeloid leukaemia, breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma such as uveal melanoma, and brain cancer such as glial brain cancer.
In any of the above aspects, when reference to“a plurality of genes or gene products” is made, it is herein meant that said plurality of genes or gene products comprises at least 2, or at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30, or at least 35, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100, or at least 150, or at least 200, or at least 250, or at least 300 of said genes or gene products. In some aspects, said plurality of genes or gene products comprises or consists of the totality of the 330 genes or gene products listed in Tables 1 and 2. In other aspects, said plurality of genes or gene products comprises or consists of the totality of the 148 genes or gene products listed in Tables 3 and 4.
In some aspects, said plurality of genes or gene products comprises or consists of the following genes or gene products:
a) Fam107a, Tex14, Trpc5, Zbtb16, Zcchc16, Adm2, Bcatl and Hhip; and/or b) Dsp, Eif2s3y, Fam107a, Flunk, Olig3, Rbfoxl and Zbtb16; and/or
c) Ifi202b, H2-Ea-ps, Klk1 b22, Gm8615, H28, H2-BI, Pyy, Gm8801 , Hist1 h2bk and LOC547349; and/or
d) Adamded , Dlgapl , Trim12a, 31 10007F17Rik, Rhox4a, Ccl21 b, Folrl , Strc and S100z; and/or
e) Rbm44, Gpr81/Hcar1 , Rec8, Sycel , Ccl8, Cled Oa, Retnla, Ccl7, Clec3b, Ccr2 and Cd209f; and/or
f) Gpx2, Klra17, Lin28b, Olfm3, Prssl , Rbm44, Rec8, Slitrk2 and Sycel .
Moreover, for the sake of conciseness, it is herein understood that expressions such as “at least one gene or gene product” or the like are not limiting, and can be interchangeably used to mean“a plurality of genes or gene products” depending on the needs and circumstances; for instance, when referring to“an inhibitor of at least one gene, or gene product derived therefrom”, the expression should be interpreted to cover an inhibitor of a plurality of genes, or gene products derived therefrom. Pharmaceutical compositions may, optionally and additionally, comprise a pharmaceutically acceptable carrier, excipient and/or diluent. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, that are physiologically compatible. Examples of suitable pharmaceutical carriers are well known in the art and include sodium chloride solutions, phosphate buffered sodium chloride solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, organic solvents and so forth. The pharmaceutically acceptable carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as e.g. phosphate, citrate, succinate, acetic acid, hyaluronic acid and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (poly)peptides, e.g., polyarginine or tripeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or non-ionic surfactants such as polysorbates, poloxamers, or PEG.
A "therapeutically effective dose" refers to that amount of active ingredient, for example a nucleic acid molecule, a polypeptide, a fusion protein, an antibody as well as a derivative or fragment(s) thereof according to the present disclosure, which ameliorates the signs or symptoms of the disease or prevent progression thereof; as would be understood in the medical arts, cure, although desired, is not required. The therapeutically effective dose of the pharmaceutical agents described herein can be estimated initially by in vitro tests, such as cell culture assays, followed by assay in model animals, usually mice, rats, rabbits, dogs, or pigs. The animal model can also be used to determine an initial preferred concentration range and route of administration.
In the frame of the present disclosure, for the sake of conciseness, the term “inhibitor” is used interchangeably with the term “antagonist”, whereas the term “activator” is used interchangeably with the term“agonist”. Also within the frame of the present disclosure, inhibitors and/or activators can be various and diverse in nature, and are particularly intended for use in the treatment of human cancer patients having a poor prognosis. Said treatments can include for instance gene therapy and/or treatment with therapeutic molecules including but not limited to antibodies, small molecules and nucleic acids such as antisense molecules.
In one aspect, the inhibitor of the invention is an antisense molecule that interferes with the activity or expression of the nucleic acid sequence (e.g. RNA) of the invention by hybridizing to the nucleic acid sequence of the corresponding nucleic acid sequence or to a variant or fragment thereof.
Examples of antisense molecules include, e.g., miRNA, siRNA, piRNA, snRNA, sh RNA or a modified antisense molecule such as GapmeRs.
The terms“microRNA,”“miRNA,” and MiR” are interchangeable and refer to endogenous or artificial non-coding RNAs that are capable of regulating gene expression. It is believed that miRNAs function via RNA interference. The terms “siRNA” and“short interfering RNA” are interchangeable and refer to single- stranded or double-stranded RNA molecules that are capable of inducing RNA interference. siRNA molecules typically have a duplex region that is between 18 and 30 base pairs in length.
The terms“piRNA” and“Piwi-interacting RNA” are interchangeable and refer to a class of small RNAs involved in gene silencing. PiRNA molecules typically are between 26 and 31 nucleotides in length.
The terms“snRNA” and“small nuclear RNA” are interchangeable and refer to a class of small RNAs involved in a variety of processes including RNA splicing and regulation of transcription factors. The subclass of small nucleolar RNAs
(snoRNAs) is also included. The term is also intended to include artificial snRNAs, such as antisense derivatives of snRNAs comprising antisense sequences directed against one or more nucleic acid sequence of the invention, or to a variant or fragment thereof.
The terms shRNA as used herein refers to a nucleic acid molecule comprising at least two complementary portions hybridized or capable of hybridizing to form a duplex structure sufficiently long to mediate RNAi (typically between 15-29 nucleotides in length), and at least one single-stranded portion, typically between approximately 1 and 10 nucleotides in length that forms a loop connecting the ends of the two sequences that form the duplex. As used herein, a GapmeR is a chimeric antisense oligonucleotide that contains a central block of deoxynucleotide monomers sufficiently long to induce RNase H cleavage. Usually, the GapmeRs of the invention are directed against one or more nucleic acid sequence of the invention, or to a variant or fragment thereof.
An "antibody" refers to an intact immunoglobulin, or to an antigen-binding portion thereof that competes with the intact antibody for specific binding to a molecular species, e.g., a polypeptide involved in (i.e. being part of) the NMDAR signalling pathway. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
Antigen-binding portions include, inter alia, Fab, Fab', F(ab')2 , Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. A Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab')2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consists of the VH and CH1 domains; a Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment consists of a VH domain.
By "bind specifically" and "specific binding" as used herein it is meant the ability of the antibody to bind to a first molecular species in preference to binding to other molecular species with which the antibody and first molecular species are admixed. An antibody is said specifically to "recognize" a first molecular species when it can bind specifically to that first molecular species. A single-chain antibody (scFv) is an antibody in which VL and VH regions are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites. One or more CDRs may be incorporated into a molecule either covalently or non-covalently to make it an immunoadhesin.
An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) non-covalently. The CDRs permit the immunoadhesin to specifically bind to a particular antigen of interest. A chimeric antibody is an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies.
An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a "bispecific" or "bifunctional" antibody has two different binding sites.
An "isolated antibody" is an antibody that 1 ) is not associated with naturally- associated components, including other naturally-associated antibodies, that accompany it in its native state, 2) is free of other proteins from the same species, 3) is expressed by a cell from a different species, or 4) does not occur in nature. It is known that purified proteins, including purified antibodies, may be stabilized with non-naturally associated components. The non-naturally-associated component may be a protein, such as albumin (e.g., BSA) or a chemical such as polyethylene glycol (PEG). A "neutralizing antibody" or "an inhibitory antibody" is an antibody that inhibits the activity of a polypeptide or blocks the binding of a polypeptide to a ligand that normally binds to it. An "activating antibody" is an antibody that increases the activity of a polypeptide.
The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is less than 1 · M, preferably less than 100 nM and most preferably less than 10 nM.
A "nucleic acid molecule" according to the present disclosure refers to a polymeric form of nucleotides and includes both sense and antisense strands of RNA (e.g. mRNA, siRNA, shRNA), cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. A "nucleic acid molecule" as used herein is synonymous with "nucleic acid" and "polynucleotide." The term "nucleic acid molecule" usually refers to a molecule of at least 10 bases in length, unless otherwise specified. The term includes single and double stranded forms of DNA. In addition, a polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
The nucleic acid molecules may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) The term "nucleic acid molecule" also includes any topological conformation, including single- stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular and padlocked conformations. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
Antisense nucleic acid compositions, or vectors that drive expression of an antisense nucleic acid, are administered to downregulate transcription and/or translation of a gene product in circumstances in which e.g. excessive production, or production of aberrant protein, is the pathophysiologic basis of disease. Antisense compositions useful in therapy can have a sequence that is complementary to coding or to noncoding regions of a gene or gene product deriving therefrom. For example, oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and + 10 from the start site, are preferred. Catalytic antisense compositions, such as ribozymes, that are capable of sequence-specific hybridization to gene transcripts, are also useful in therapy.
In a preferred aspect, the antisense is an oligonucleotide (e.g. an antisense molecule as described herein) having a sequence designed to hybridize with a nucleic acid sequence of a gene, or gene product such as an mRNA deriving therefrom, selected from a list including:
a. at least one member of the genes listed in Tables 2 and/or 4; and/or
b. the NMDA receptor gene; and/or
c. a member of the NMDA receptor signalling pathway; and/or
d. a member of the DLGAP family of genes; and/or e. the HSF1 gene; and/or
f. the FMR1 gene
as well as a fragment, an allelic variant, a homolog and/or a substantially similar or hybridizing nucleic acid of the foregoing.
Still a further object of the subject-matter described in this disclosure relates to a kit for in vitro analysis aimed at determining the prognosis of a patient having a cancer, said cancer being altered in the NMDAR signalling pathway, said kit comprising a reagent that selectively interacts with one or more of:
(a) at least one of a plurality of gene products of Tables 1 to 4; and/or
(b) a nucleic acid molecule having at least 95% sequence identity to a gene product derived from (a); and/or
(c) a polypeptide encoded by a nucleic acid molecule of (b); and/or
(d) a polypeptide comprising an amino acid sequence with at least 95% sequence identity to a polypeptide of (b).
The kit of the invention may also comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The label or package insert may comprise instructions for use thereof. Instructions included may be affixed to packaging material or may be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and
communicating them to an end user is contemplated by this disclosure. The cancer can be selected from a list comprising blood cancer such as acute myeloid leukaemia, breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma such as uveal melanoma, and brain cancer such as glial brain cancer.
Another aspect of the subject matter described in the present specification relates to a method of treatment of a cancer in a patient being diagnosed to have a poor prognosis, comprising administering:
a. an inhibitor of at least one gene, or gene product derived therefrom, listed in Tables 2 and/or 4; and/or
b. at least one gene, or gene product derived therefrom, listed in Tables 1 and/or 3, or an activator thereof; and/or
c. an inhibitor of the NMDA receptor; and/or
d. an inhibitor of a gene product member of the NMDA receptor signalling pathway; and/or
e. an inhibitor of a member of the DLGAP family of genes, or of gene product(s) derived therefrom; and/or
f. an inhibitor of the HSF1 gene, or of gene product(s) derived therefrom; and/or
g. an inhibitor of the FMR1 gene, or of gene product(s) derived therefrom. Said inhibitors or activators can be selected from a non-exhaustive list comprising a nucleic acid molecule, a polypeptide, a fusion protein, an antibody as well as a derivative, variant or fragment(s) thereof, as described herein.
In one aspect, said inhibitor is selected from a group including but not limited to MK801 , memantine, Ifenprodil, ketamine and Ro 25-6981.
Preferably, the method of treatment described above follows the method for determining the prognosis for survival of a patient having a cancer of the invention. A further object of the subject-matter described in this disclosure relates to the use of a pharmaceutical composition of the invention in the preparation of a medicament for the treatment of a cancer in a patient being diagnosed to have a poor prognosis. "Administering", as it applies in the present invention, refers to contact of an effective amount of an inhibitor or activator of the invention, to the patient.
In general, methods of administering a composition comprising an inhibitor or activator that is in the form of a miRNA, siRNA, piRNA, hnRNA, snRNA, esiRNA, shRNA, or antisense oligonucleotide are well known in the art. In particular, the routes of administration already in use for nucleic acid therapeutics, along with formulations in current use, provide preferred routes of administration and formulation for the nucleic acids described herein.
Nucleic acid compositions can be administered by a number of routes including, but not limited to: oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, intra- or transcranial or rectal means. Nucleic acids can also be administered via gene delivery vectors or nanoparticules. Such administration routes and appropriate formulations are generally known to those of skill in the art. As used herein, a gene delivery vector, preferably in the form of a plasmid or a vector, comprises one or more nucleic acid(s) encoding an inbitor or activator of the present invention. As used herein, a "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Suitable vectors include derivatives of SV40 and known bacterial plasmids, e. g.,
E. coli plasmids col El, pCRI, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e. g., the numerous derivatives of phage X, e. g., NM989, and other phage DNA, e. g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2* plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
Various viral vectors are used for delivering nucleic acid to cells in vitro or in vivo. Non-limiting examples are vectors based on Herpes Viruses, Pox- viruses, Adeno- associated virus, Lentivirus, and others. In principle all of them are suited to deliver the expression cassette comprising an expressible nucleic acid molecule that codes for an inbitor or activator of the present invention. In a preferred aspect, said viral vector is an adenoviral vector, preferably a replication competent adenovirus.
Methods of administering a composition comprising an inhibitor or activator that is in the form of a polypeptide, a fusion protein, an antibody as well as a derivative, variant or fragment(s) thereof, as described herein are also well known in the art.
Administration of the formulations (e.g. pharmaceutical compositions) described herein may be accomplished by any acceptable method which allows the inhibitor or activator of the invention to reach its target. The particular mode selected will depend of course, upon factors such as the particular formulation, the severity of the state of the subject being treated, and the dosage required for therapeutic efficacy. The actual effective amounts of drug can vary according to the specific drug or combination thereof being utilized, the particular composition formulated, the mode of administration, and the age, weight, condition of the patient, and severity of the symptoms or condition being treated.
Any acceptable method known to one of ordinary skill in the art may be used to administer a formulation to the subject. The administration may be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the cancer being treated. Injections can be e.g., intravenous, intradermal, subcutaneous, intra- or
transcranial, intramuscular, or intraperitoneal.
The injections can be given at multiple locations. Implantation includes inserting implantable drug delivery systems, e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol matrixes, polymeric systems, e.g., matrix erosion and/or diffusion systems and non-polymeric systems, e.g., compressed, fused, or partially-fused pellets. Inhalation includes administering the composition with an aerosol in an inhaler, either alone or attached to a carrier that can be absorbed. For systemic administration, it may be preferred that the composition is
encapsulated in liposomes.
Preferably, the antisense nucleic acid compositions, or vectors or plasmids that drive expression of an inhibitor or activator of the invention (such as for example an antisense nucleic acid), are provided in a manner which enables tissue-specific uptake of the nucleic acid delivery system. Techniques include using tissue or organ localizing devices, such as wound dressings or transdermal delivery systems, using invasive devices such as vascular or urinary catheters, and using interventional devices such as stents having drug delivery capability and
configured as expansive devices or stent grafts.
The formulations may be delivered using a bio-erodible implant by way of diffusion or by degradation of the polymeric matrix.
The administration of the formulation may be designed so as to result in sequential exposures to an inhibitor or activator of the invention, or the pharmaceutical composition comprising the same, over a certain time period, for example, hours, days, weeks, months or years. This may be accomplished, for example, by repeated administrations of a formulation or by a sustained or controlled release delivery system in which the inhibitor or activator of the invention or the
pharmaceutical composition comprising the same, is delivered over a prolonged period without repeated administrations. Administration of the formulations using such a delivery system may be, for example, by oral dosage forms, bolus injections, transdermal patches or subcutaneous implants. Maintaining a
substantially constant concentration of the composition may be preferred in some cases.
Other delivery systems suitable include, but are not limited to, time-release, delayed release, sustained release, or controlled release delivery systems. Such systems may avoid repeated administrations in many cases, increasing
convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include, for example, polymer-based systems such as polylactic and/or polyglycolic acids, polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides,
polyorthoesters, polyhydroxybutyric acid, and/or combinations of these.
Microcapsules of the foregoing polymers containing nucleic acids are described in, for example, U.S. Patent No. 5,075, 109. Other examples include nonpolymer systems that are lipid-based including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; liposome-based systems; phospholipid based-systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants. Specific examples include, but are not limited to, erosional systems in which the inhibitor or activator is contained in a formulation within a matrix (for example, as described in U.S. Patent Nos. 4,452,775, 4,675, 189, 5,736,152, 4,667,013, 4,748,034 and
5,239,660), or diffusional systems in which an active component controls the release rate (for example, as described in U.S. Patent Nos. 3,832,253, 3,854,480, 5,133,974 and 5,407,686). The formulation may be as, for example, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems. The system may allow sustained or controlled release of the composition to occur, for example, through control of the diffusion or erosion/degradation rate of the formulation containing the inhibitor or activator of the invention. In addition, a pump-based hardware delivery system may be used for delivery. Examples
1. Dlgap 1/GKAP is the most differentially expressed candidate modifier gene when comparing the invasive B6 and non-invasive C3H backgrounds
Initially, the inventors further mined the expression data from Chun et al. to ascertain the ratio of gene expression in both normal pancreatic islets (consisting mostly of b cells) and fully developed b cell tumours (PanNETs) from B6 and C3H mice. Strikingly, dlgapl (encoding GKAP protein) was the most differentially expressed gene in the locus, both in normal pancreatic islets and in PanNETs (Figure 9A), and its expression remained stable throughout different stages of tumour progression. Consistently with this finding, when examined the GKAP transcripts in a broad panel of tissues obtained from wild-type B6 and C3H backgrounds, elevated GKAP expression in a number of B6 tissues compared to C3H tissues, in various organs, was found (Figure 9B).
In order to investigate expression and potential functional roles of GKAP in further depth, PanNET cell lines from each of the two strains were derived; qRT-PCR showed a clear difference in GKAP transcripts, with higher levels in · TC-B6 than in •TC-C3H (Figure 1A), a result consistent with the qRT-PCR analysis of FACS- purified constituent cell types from primary PanNET tumours (Figure 1 B).
It was then investigated the basis for the elevated expression of GKAP in B6 PanNET tumours, focusing on potential regulatory polymorphisms, given that there were no differences in GKAP protein sequence. Genomic analysis for single nucleotide polymorphisms (SNP) in putative transcription factor-binding sites within 5Kb up- and down-stream of the GKAP/dlgap1 gene identified a SNP (rs33397766) that mapped to a potential Heat Shock Factor (HSF) binding element (Figure 1 C, upper panel). The consensus HSF1 binding site is TTCnnGAAnnTTC (10, SEQ ID No. 3), in which the TTC repeats at each end are usually highly conserved, whereas the GAA site can be variable, as shown in several studies. Surprisingly, while the B6 version of the GKAP gene has TTC repeats on both ends (SEQ ID No. 1), and therefore is predicted to retain HSF1 binding capacity, in the C3H version of the GKAP gene the TTC repeat at one end is missing (SEQ ID No. 2), becoming TCC due to the SNP variation, which may impair HSF1 binding. Interestingly, HSF1 has been previously identified as a modifier of cancer progression, via a transcriptional program independent of heat shock.
To determine whether the proposed binding element in the B6 GKAP gene region was indeed differentially bound by HSF1 in B6 and C3H, a ChIP assay was performed with an anti-HSF1 antibody, using the aforementioned bTOBb and bTO C3H mPanNET cell lines. As expected, a two-fold enrichment of the HSF1 binding at the proposed site within the GKAP gene in B6 was observed when compared to C3H (Figure 1 C, lower panel). Furthermore, GKAP expression was decreased when we knocked down HSF1 in bTO-B6 cells (Figure 1 D), suggesting that HSF1 is an upstream regulator of GKAP. Notably, the levels of HSF1 protein were not significantly different between B6 and C3H mPanNETs, suggesting that differential binding of HSF-1 protein to the SNP site in the GKAP gene was responsible for the differential expression.
2. Cancer cells from the invasive B6 background have higher NMDAR pathway activity than those from the non-invasive C3H background
By using a previously established invasion assay mimicking interstitial pressure- driven fluid flow to induce autocrine secretion of the glutamate ligand of NMDAR, which activates NMDAR signalling and increases invasiveness in a cancer cell line, B6 cancer cells (* TC-B6) were found to be more invasive than C3H cancer cells (bTO03H) (Figure 1 E). Notably, under flow conditions glutamate secretion was significantly increased in * TC-B6 compared to * TC-C3H (Figure 1 F) compared to static conditions. Moreover, when applied NMDA to cultured cancer cells in ‘puffs’ from a micropipette, a strong signal for calcium influx was observed in * TC-B6 cells, whereas no signal was detectable in * TC-C3H (Figures 2A and 2B), indicating the presence of functional NMDA-responding calcium channels (i.e., NMDAR) in bTO- B6, but not in bTO03H.
3. GKAP expression is associated with differential NMDAR activity and sensitivity to pharmacological inhibition in tumours
The NMDAR signalling pathway includes the NMDAR subunits GluN1 (Grinl) and GluN2b (Grin2b), the key scaffold proteins PSD-95 (Dlg4) and GKAP (Dlgapl), and the principal glutamate transporters vGlutl (Sid 7a8) and vGlut2 (Sid 7a7). By qRT- PCR, the inventors found that all of these genes were expressed in both B6 and C3H PanNETs at comparable levels, except for GKAP (Dlgapl), which was significantly lower in the less invasive C3H background (Figure 3A, B). Notably, while GluN2b was expressed at similar levels in PanNETs from both strain backgrounds (Figure 3B, 3C middle panel), the phosphorylated form of GluN2b (GluN2b pY1252), an indicator of NMDAR activation, was only detectable in B6 but not in C3H PanNETs (Figure 3C, right panel). As anticipated by the absence of NMDAR activity (reflected by no detectable pGluN2b), C3H RIP1Tag2 mice were largely non-responsive in a pharmacological“intervention” trial using the NMDA receptor inhibitor MK801 , in contrast to B6 RIP1Tag2 mice that are responsive (Figure 3D). The result establishes that, despite similar expression levels of NMDAR mRNA and protein in PanNETs from both genetic backgrounds, comparatively elevated expression of the scaffold protein GKAP in B6 PanNETs was associated with higher signalling activity, and hence increased sensitivity to pharmacological inhibition. 4. GKAP knockdown phenocopies the“iow-NMDAR activity” status and reduces • TC-3 invasiveness in vitro
The qRT-PCR data implied that the comparatively higher levels of GKAP in B6 cancer cells might be sufficient for activation of glutamate secretion and NMDAR signalling, as GKAP/dlgap1 was the only differentially expressed gene from the core NMDAR signalling axis (Figure 3A). Furthermore, the“strain signature” from the tumour RNA-seq data implicated GKAP to be one of the defining genes for B6 vs. C3H PanNETs (Figure 7A). It was therefore evaluated shRNA knockdown (Openbiosystem, Clone ID: TRCN0000088935. )of GKAP in cultured PanNET cancer cells. Notably, GKAP has nine protein-coding splice variants, of which three were differentially expressed in B6 vs C3H PanNETs (Figure 1 1 B) (the remaining six variants were all below the detection threshold). Accordingly, a shRNA knockdown design that targeted all three of these GKAP isoforms was selected. The GKAP-knockdown cells showed decreased GluN2b phosphorylation, indicative of reduced NMDAR signalling activity (Figure 4A). Furthermore, GKAP-knockdown cells exhibited a significantly reduced response to the NMDA ligand (Figure 4B). Importantly, · TC-3 invasiveness under flow conditions was also decreased by the knockdown (Figure 4C). Thus, the knockdown of GKAP mRNA functionally phenocopied the C3H phenotype, in relation to NMDAR activity and cancer cell invasiveness.
5. NMDAR pathway activity modulates several regulators of the translational machinery
Having determined that differential expression of GKAP governed NMDAR signalling activity-mediated cancer cell invasiveness, the inventors sought to uncover potential downstream invasion effector mechanisms. Since no obvious invasion pathways were associated with the strain and MK801 -inhibited signatures from PanNETs, the GKAP control and knockdown cell lines was further characterized, looking for differentially affected biological processes. In particular, protein translation activities were targeted, because NMDAR activity has been shown to be crucial for mRNA localization and protein translation in neurons.
Although FMRP was not identified as a driver in the signature analysis of the RNA- seq datasets, higher FMRP protein expression in B6 tumours compared to C3H tumours was found (Figure 10B); moreover, in B6 mPanNETs, FMRP expression was particularly elevated in the more invasive tumours (Figure 4D, 10C, 10D), and in liver metastases (Figure 4D). In vitro, inhibiting NMDAR pathway activity either by MK801 treatment or GKAP knockdown decreased FRMP protein expression (Figures 4E and 4F).
In light of the observation that NMDAR signalling (through GKAP) regulates translational machinery, the inventors asked whether targeting the NMDAR pathway affected HSF1 activation, known to be a major target downstream of deregulated translational activity in cancer cells. Indeed, HSF1 phosphorylation at serine 326, a marker for activated HSF1 transcription activity, was decreased by both MK801 treatment and GKAP knockdown in - TC-3 (Figure 4F), identifying HSF1 as a regulatory target of NMDAR signalling. Collectively, the data presented above establishes GKAP as a polymorphic modifier gene that differentially regulates an invasive growth program mediated by NMDAR signalling as a function of differential expression governed by genetic background.
6. NMDAR activity in pancreatic ductai adenocarcinoma ( PDAC )
To investigate the potential generality of these results, asking whether NMDAR activation through GKAP and the consequent up-regulation of FMRP/HSF1 could be important for other cancer types, the inventors then focused on an appraisal of pancreatic ductal adenocarcinoma (PDAC), since NMDAR expression was previously implicated in both mouse and human PDAC. In tumours from the PDAC genetically engineered mouse model (GEMM), the biomarkers p-GluN2b, GKAP, HSF1 and FMRP were all highly expressed both in the primary tumours and in liver metastases (Figure 6A), suggesting that the NMDAR/GKAP/HSF1/FMRP pathway is active in mPDAC. Furthermore, in human tissue microarrays (TMAs) of PDAC, GluN2b activity was elevated, as was expression of GKAP, HSF1 and FMRP in the progression from premalignant lesions to primary pancreatic ductal adenocarcinomas to lymph node-metastases (Figure 6B i, ii). Notably, active NMDAR signalling (as indicated by p-GluN2b expression) also positively correlated with HSF1 and FMRP expression and with larger tumour size in human PDAC (Figure 6B iii). Furthermore, human tumours that showed vascular invasion, an independent poor prognostic factor associated with liver metastasis, expressed higher p-GluN2b compared to tumours without vascular invasion (Figure 6B iv). This evidence collectively supports the proposition that NMDAR activity is contributing to human PDAC malignancy.
Since both GKAP and GluN2b were detected in mPDAC tumours, cell lines from primary mPDAC tumours were generated for functional perturbation. Two lines were selected for the following study, 4361 and 2263, representative of high/low GKAP protein expression respectively. Western blot analysis revealed that the high GKAP- expressing 4361 line had elevated GluN2b phosphorylation compared to the low GKAP-expressing line (Figure 5A, left panel). Consistent with previous observation in PanNET, the comparatively higher GKAP expression in the 4361 cell line correlated with increased invasiveness, especially under flow conditions (Figure 5A, right panel), as well as with increased sensitivity to MK801 inhibition (Figures 5B). Concordantly, knocking down GKAP with shRNA (Openbiosystem, Clone ID: TRCN0000088935.) diminished GluN2b phosphorylation and dampened the invasion of the 4361 cell line (Figure 5C i). The link between GKAP expression, NMDAR pathway activity, and cancer cell invasiveness was further validated in a human PDAC cell line, DanG, which were previously reported to have high GluN2b expression, and to be highly invasive in flow-stimulated invasion assays as well as sensitive to MK801 inhibition. Congruently, when GKAP was knocked down using siRNA (Thermo Fisher Scientific, #4392420, Assay ID s17644, s52141 ), the flow- enhanced invasiveness of human DanG PDAC cells was concomitantly decreased (Figure 5C ii).
To explore whether, similarly to mice, a NMDAR/GKAP/HSF1/FMRP axis exists also in human PDAC, inventors treated DanG and SUIT2, with MK801 , which decreased HSF1 phosphorylation and FMRP expression (Figure 5D). Finally, knocking down either FMRP or HSF1 in hPDAC cells decreased cancer cell invasiveness in the flow-guided invasion assay (Figure 5E), establishing that FMRP and HSF1 are functionally important for invasion.
7. Transcriptome profiling reveals genetic background-specific signatures affected by inhibition of NMD AR signaling in mPanNETs
The data in Figures 3C and 3D indicated that B6 control tumours were NMDAR pathway-active, whereas both C3H control tumours and MK801 -treated B6 tumours were NMDAR pathway-inactive. It was therefore analysed RNA-seq data from B6 control tumours, B6 MK801 -treated tumours, and C3H control tumours, seeking to identify gene expression signatures (Biton et al. 2013; Miettinen et al. 2017; Rutledge and Bouveresse 2013) corresponding to these phenotypes (Figure 7A). Signature correlation values were represented as z-scores (centred and scaled to yield mean = 0 and standard deviation = 1 ). The higher a |Z| score is, the more likely that the gene is a major factor (driver) in the signature. In this analysis, genes with |Z|>2.5 were considered to be significantly associated with a corresponding signature.
First, it was identified a“strain signature” that distinguished untreated B6 tumour samples from C3H tumour samples (Figure 7Bi,ii; Table 1 ). Notably, amongst all the differentially expressed genes between the two strain backgrounds, dlgap1/GKAP (|Z|= 9.6) was the second most poorly expressed signature gene in C3H. Next, a set of genes was identified that distinguished MK801 -treated B6 tumours from untreated B6 tumours, named“MK801 treatment signature” (Figure 7C; Table 2). In genes upregulated by MK801 treatment, the top driver gene was FAM107a (family with sequence similarity 107, also known as DRR1 ) (|Z|= 1 1 .72), implicated as a tumour suppressor in renal cell carcinoma (Yamato T et al., 1999; Wang et al., 2000), and reportly down-regulated in neuroblastoma (Asano et al., 2010), a tumour where NMDAR signalling was shown to be activated (North et al., 1997).
Next, a gene set enrichment analysis (GSEA, Subramanian et al., 2005) between the MK801 - treatment signature and the B6-C3H strain signature revealed that genes elevated by MK801 inhibition of NMDAR signalling also show high expression in C3H control samples, and vice versa (not shown), suggesting that C3H tumors were similar to MK801 -treated B6 tumors when compared to B6 control tumors, and in support to the hypothesis that differences in NMDAR pathway activity are indeed one of the phenotype-defining features between the B6 and C3H PanNETs.
Having confirmed the high similarity between the strain signature and the MK801 treatment signature, the inventors aimed at identifying a common (core) gene expression profile for the NMDAR pathway-low phenotype. Through leading edge analysis of these two datasets, looking for common driver genes involved in both signatures (high in B6 control, low in both MK801 -treated and C3H samples, or vice versa), the inventors identified a total of 148 genes, and named this signature as the “NMDAR-pathwayl0W gene/NMDAR pathway-low signature” (Figure 7D; Tables 3 and 4).
Pathway analysis of the “NMDAR-pathwaylow gene signature” identified “Neurogenesis and Synaptogenesis” to be one of the major pathways involving these genes (Figure 1 1 A), consistent with the interpretation that these genes are regulated by NMDAR signalling. Notably, one of the major phenotypes of B6 MK801 -treated tumours is decreased proliferation hence low tumour burden, in contrast to C3H tumours (Figure 7A); therefore, applying these selection criteria allows us to exclude common genes associated with proliferation, and focus on genes specific reflecting NMDAR pathway activation.
The effects of MK801 treatment on the malignant phenotype of PDAC were further evaluated in experimental therapeutic trials performed in the PDAC GEMM (LSL- KrasG12D; p53LSLR172H; p48Cre) using the pharmacological inhibitor MK801. The data revealed that MK801 conveyed a survival benefit, which was likely limited by the toxicity of MK801 : mice in the MK801 treatment group suffered from severe weight loss, and had to be sacrificed despite lacking significant tumour burden at end stage. Notably, a survival benefit was also observed using a clinically approved, albeit less potent NMDAR inhibitor, memantine (Figure 8A).
8. Transcriptome signatures identified in mPanNETs can predict prognosis in various human cancer types
The“MK801 treatment signature”, identified from the aforementioned mPanNET RNA-seq data analysis (Figure 7C), was applied to query the TCGA cancer patient database for associated survival differences. A remarkable and significant survival benefit was associated with PDAC patients whose tumours correlated with the “MK801 -treatment signature” - as if tumours from these patients had been treated with MK801 - compared to the rest of the patients (Figure 8B); moreover, lower grade tumours (G1 , G2) showed a more pronounced association with the MK801 treatment signature compared to higher grade tumours (G3, G4)(Figure 12A). It was found that the“MK801 treatment signature” is a significant, independent prognostic factor, even surpassing the predictive power of T and N stages (Figure 8C).
Strikingly, in addition to PDAC, this “MK801 treatment signature” was also significantly associated with favourable prognosis in patients from several other cancer types, including glial brain cancers, kidney cancers, uveal melanoma, and acute myeloid leukaemia (Figure 8D). Interestingly, it was found that amongst glial brain cancers, low grade glioma tumours were significantly more correlated with the MK801 treatment/pathway-low signature when compared to advanced (more invasive and aggressive) glioblastoma tumours (Figure 8E), suggestive of the latter’s dependence on NMDAR pathway activity to drive the highly malignant phenotype.
In order to test if the “NMDAR-pathwayl0W gene signature” has prognostic importance, inventors analysed the same patient tumour dataset using this “NMDAR-pathwayl0W gene signature” gene set, and found that the 148 common driver genes could also predict survival in PDAC patients (Figure 8F) as well as a few other cancer types (Figure 8G, 12C), comparable to the association revealed by the 330 genes in the full “MK801 treatment signature”. Interestingly, in uveal melanoma patients, the“NMDAR-pathwayl0W gene signature” showed even better separation of the survival curves compared to the “MK801 treatment signature” (Figure 8G, 12B).
Importantly, these associations indicate that the MK801 -treatment signature (and its key constituents - the“NMDAR-pathwayl0Wgene signature”) is not specific to mouse PanNET, where it was discovered, and suggest that the signature has a prognostic potential for certain human cancers. In sum, these data begin to broaden the association of NMDAR signalling via HSF-1 and FMRP with invasive tumour growth and malignancy. The results additionally suggest that NMDAR antagonists may be therapeutically beneficial in PDAC (and other selected cancer types) patients whose tumours are inferred to have elevated NMDAR signalling by virtue of lacking this “NMDAR-low” MK801 -treatment signature. n Table 1 : Genes/gene products Up-regulated by MK801 Treatment
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List of References
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2. Sadanandam A, Wullschleger S, Lyssiotis CA, Grotzinger C, Barbi S, Bersani S, et al. A Cross-Species Analysis in Pancreatic Neuroendocrine Tumors Reveals Molecular Subtypes with Distinctive Clinical, Metastatic, Developmental, and Metabolic Characteristics. Cancer Discov. 2015;5:1296-313.
3. Asano Y, Kishida S, Mu P, Sakamoto K, Murohara T, Kadomatsu K. DRR1 is expressed in the developing nervous system and downregulated during neuroblastoma carcinogenesis. Biochem Biophys Res Commun. 2010;394:829-35.
4. North WG, Fay MJ, Du J, Cleary M, Gallagher JD, McCann FV. Presence of functional NMDA receptors in a human neuroblastoma cell line. Mol Chem Neuropathol. 1997;30:77-94.
5. Li L, Hanahan D. Hijacking the neuronal NMDAR signaling circuit to promote tumor growth and invasion. Cell. 2013;153:86-100.
6. Abbott, L. F., and Nelson, S. B. (2000). Synaptic plasticity: taming the beast. Nat Neurosci 3 Suppl, 1 178-1 183.
7. Yamato T, Orikasa K, Fukushige S, Orikasa S, Horii A. Isolation and characterization of the novel gene, TU3A, in a commonly deleted region on 3p14.3- ->p14.2 in renal cell carcinoma. Cytogenet Cell Genet. 1999;87:291 -5.
8. Wang L, Darling J, Zhang JS, Liu W, Qian J, Bostwick D, et al. Loss of expression of the DRR 1 gene at chromosomal segment 3p21.1 in renal cell carcinoma. Genes Chromosomes Cancer. 2000;27:1 -10. 9. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545-50.
10. Biton, A., Zinovyev, A., Barillot, E., and Radvanyi, F. (2013). MineICA: Independent component analysis of transcriptomic data (www.bioconductor.org).
11. Miettinen, J., Nordhausen, K. and Taskinen, S. (2017), Blind Source Separation Based on Joint Diagonalization in R: The Packages JADE and BSSasymp, Journal of Statistical Software, 76(2).
12. Rutledge, D. N., and Jouan-Rimbaud Bouveresse, D. (2013). Independent Components Analysis with the JADE algorithm. TrAC Trends in Analytical Chemistry 50, 22-32.

Claims

Claims
1. A method for determining the prognosis for survival of a patient having a cancer, said cancer being altered in the NMDAR signalling pathway, the method comprising a step of determining the transcription level and/or the expression level and/or the activity of a plurality of genes or gene products selected from genes listed in Table 1 to 4 in a sample from said cancer patient relative to a control, wherein the differential transcription and/or expression and/or activity of said plurality of genes or gene products relative to a control is indicative of cancer aggressiveness and therefore of the patient’s prognosis.
2. The method according to claim 1 , wherein the differential transcription and/or expression and/or activity of said plurality of genes or gene products relative to a control is up-regulation or down-regulation of transcription and/or expression and/or activity of said genes or gene products compared to a control level.
3. The method according to claim 2, wherein down-regulation of a plurality of genes or gene products selected from genes listed in Table 1 and/or 3, and/or up- regulation of a plurality of genes or gene products selected from genes listed in Table 2 and/or 4, correlates with a high activation of the NMDAR signalling pathway in a cancer and with a poor prognosis, whereas up-regulation of a plurality of genes or gene products selected from genes listed in Table 1 and/or 3, and/or down- regulation of a plurality of genes or gene products selected from genes listed in Table 2 and/or 4 correlates with a low activation of the NMDAR signalling pathway in a cancer and with a favourable prognosis.
4. The method of anyone of the preceding claims, wherein the gene product is a polypeptide or a nucleic acid.
5. The method of anyone of the preceding claims, wherein said plurality of genes or gene products comprises or consists of the following genes or gene products:
a) Fam107a, Tex14, Trpc5, Zbtb16, Zcchc16, Adm2, Bcatl and Hhip; and/or b) Dsp, Eif2s3y, Fam107a, Flunk, Olig3, Rbfoxl and Zbtb16; and/or
c) Ifi202b, H2-Ea-ps, Klk1 b22, Gm8615, H28, H2-BI, Pyy, Gm8801 , Hist1 h2bk and LOC547349; and/or
d) Adamded , Dlgapl , Trim 12a, 31 10007F17Rik, Rhox4a, Ccl21 b, Folrl , Strc and S100z; and/or
e) Rbm44, Gpr81/Flcar1 , Rec8, Sycel , Ccl8, Cled Oa, Retnla, Ccl7, Clec3b, Ccr2 and Cd209f; and/or
f) Gpx2, Klra17, Lin28b, Olfm3, Prssl , Rbm44, Rec8, Slitrk2 and Sycel .
6. The method of anyone of the preceding claims, wherein the genes or gene products whose up-regulation or over-expression is associated with a good prognosis, comprise or consist of:
a) Fam107a, Tex14, Trpc5, Zbtb16, Zcchc16, Adm2, Bcatl and Hhip; and/or b) Dsp, Eif2s3y, Fam107a, Flunk, Olig3, Rbfoxl and Zbtb16; and/or
c) Ifi202b, H2-Ea-ps, Klk1 b22, Gm8615, H28, H2-BI, Pyy, Gm8801 , Hist1 h2bk and LOC547349.
7. The method of anyone of the preceding claims, wherein the genes or gene products whose down-regulation or down-expression is associated with a good prognosis comprise or consist of:
d) Adamded , Dlgapl , Trim12a, 31 10007F17Rik, Rhox4a, Ccl21 b, Folrl , Strc and S100z; and/or
e) Rbm44, Gpr81/Flcar1 , Rec8, Sycel , Ccl8, Cled Oa, Retnla, Ccl7, Clec3b, Ccr2 and Cd209f; and/or f) Gpx2, Klra17, Lin28b, Olfm3, Prssl , Rbm44, Rec8, Slitrk2 and Sycel .
8. The method of anyone of the preceding claims, wherein the cancer is selected from the group comprising blood cancer (such as acute myeloid leukaemia), breast cancer, ovarian cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, and brain cancer.
9. A method for improving the prognosis for a cancer patient being diagnosed to have a poor prognosis, comprising modulating the transcription level and/or the expression level and/or the activity of:
- at least one of a plurality of genes or gene products listed in Tables 1 to 4; and/or
- a gene or gene product member of the NMDA receptor signalling pathway; and/or
- a gene or gene product member of the DLGAP family of genes; and/or
- a HSF1 gene or gene product; and/or
- a FMR1 gene or gene product.
10. The method according to claim 9, wherein modulating transcription level and/or the expression level and/or the activity of genes or gene products comprises increasing the transcription level and/or the expression level and/or the activity of genes or gene products whose up-regulation or over-expression is associated with a good prognosis.
1 1. The method according to claim 10, wherein the genes or gene products are selected from a group comprising the genes or gene products listed in Tables 1 and/or 3.
12. The method according to claim 10, wherein modulating transcription level and/or the expression level and/or the activity of genes or gene products comprises decreasing the transcription level and/or the expression level and/or the activity of genes or gene products whose down-regulation or down-expression is associated with a good prognosis.
13. The method according to claim 12, wherein the genes or gene products are selected from a group comprising the genes or gene products listed in Tables 2 and/or 4.
14. The method according to claims 9 to 13, wherein said modulation is carried out by administering to a cancer patient:
a) an inhibitor of at least one gene, or gene product derived therefrom, listed in Tables 2 and/or 4; and/or
b) at least one gene, or gene product derived therefrom, listed in Tables 1 and/or 3, or an activator thereof; and/or
c) an inhibitor of the NMDA receptor; and/or
d) an inhibitor of a gene product member of the NMDA receptor signalling pathway; and/or
e) an inhibitor of a member of the DLGAP family of genes, or of gene product(s) derived therefrom; and/or
f) an inhibitor of the HSF1 gene, or of gene product(s) derived therefrom; and/or g) an inhibitor of the FMR1 gene, or of gene product(s) derived therefrom.
15. The method according to claim 14, wherein said inhibitor or activator is selected from the group comprising a nucleic acid molecule, a polypeptide, a fusion protein, an antibody as well as a derivative or fragment(s) thereof.
16. The method according to claim 14, item c, wherein said inhibitor is selected from a group comprising MK801 , memantine Ifenprodil, ketamine and Ro 25-6981 or a combination thereof.
17. The method according to claim 14, wherein said inhibitor of at least one gene, or gene product derived therefrom, is an antisense molecule that interferes with the activity or expression of a nucleic acid sequence or to a variant or fragment thereof.
18. The method according to claim 17, wherein said antisense molecule is selected from the group comprising an miRNA, siRNA, piRNA, snRNA, shRNA or a modified antisense molecule (such as GapmeRs).
19. A pharmaceutical composition for use in the treatment of a cancer in a patient being diagnosed to have a poor prognosis, comprising:
a) an inhibitor of at least one gene, or gene product derived therefrom, listed in Tables 2 and/or 4; and/or
b) at least one gene, or gene product derived therefrom, listed in Tables 1 and/or 3, or an activator thereof; and/or
c) an inhibitor of the NMDA receptor; and/or
d) an inhibitor of a gene product member of the NMDA receptor signalling pathway; and/or
e) an inhibitor of a member of the DLGAP family of genes, or of gene product(s) derived therefrom; and/or
f) an inhibitor of the HSF1 gene, or of gene product(s) derived therefrom; and/or g) an inhibitor of the FMR1 gene, or of gene product(s) derived therefrom.
20. The pharmaceutical composition for use according to claim 19, item c, wherein said inhibitor is selected from a group comprising MK801 , memantine, Ifenprodil, ketamine and Ro 25-6981 or a combination thereof.
21. The pharmaceutical composition for use according to claim 19, wherein said inhibitor of at least one gene, or gene product derived therefrom, is an antisense molecule.
22. The pharmaceutical composition for use according to claim 21 , wherein said antisense molecule is selected from the group comprising an miRNA, siRNA, piRNA, snRNA, sh RNA or a modified antisense molecule (such as GapmeRs).
23. A kit for in vitro analysis aimed at determining the prognosis of a patient having a cancer, said cancer being altered in the NMDAR signalling pathway, said kit comprising a reagent that selectively interacts with one or more of:
a) at least one of a plurality of gene products of Tables 1 to 4; and/or
b) a nucleic acid molecule having at least 95% sequence identity to a gene product derived from (a); and/or
c) a polypeptide encoded by a nucleic acid molecule of (b); and/or
d) a polypeptide comprising an amino acid sequence with at least 95% sequence identity to a polypeptide of (b).
24. A method of treatment of a cancer in a patient being diagnosed to have a poor prognosis, comprising administering:
a. an inhibitor of at least one gene, or gene product derived therefrom, listed in Tables 2 and/or 4; and/or
b. at least one gene, or gene product derived therefrom, listed in Tables 1 and/or 3, or an activator thereof; and/or
c. an inhibitor of the NMDA receptor; and/or d. an inhibitor of a gene product member of the NMDA receptor signalling pathway; and/or
e. an inhibitor of a member of the DLGAP family of genes, or of gene product(s) derived therefrom; and/or
f. an inhibitor of the HSF1 gene, or of gene product(s) derived therefrom; and/or
g. an inhibitor of the FMR1 gene, or of gene product(s) derived therefrom.
25. The method according to claim 24, wherein said inhibitor or activator is selected from the group comprising a nucleic acid molecule, a polypeptide, a fusion protein, an antibody as well as a derivative or fragment(s) thereof.
26. The method according to claim 24, item c, wherein said inhibitor is selected from a group comprising MK801 , memantine Ifenprodil, ketamine and Ro 25-6981 , or a combination thereof.
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