阅读说明：本技术 使用靶基因表达的数学建模评估mapk-ap-1细胞信号传导途径活性 (Assessment of MAPK-AP-1 cell signaling pathway activity using mathematical modeling of target gene expression ) 是由 A·范德斯托尔佩 L·H·F·M·霍尔特泽尔 于 2018-09-28 设计创作，主要内容包括：本发明涉及用于基于在对象样品中测量的MAPK-AP-1细胞信号传导途径的三个或更多个靶基因的表达水平来推测对象中MAPK-AP-1细胞信号传导途径活性的计算机执行方法。本发明进一步涉及用于推测对象中MAPK-AP-1细胞信号传导途径活性的装置、非暂时性存储介质和计算机程序。本发明进一步涉及用于测量对象样品中MAPK-AP-1细胞信号传导途径的三个或更多个靶基因的表达水平的试剂盒,用于推测对象中MAPK-AP-1细胞信号传导途径活性的试剂盒,以及这些试剂盒在进行所述方法中的应用。(The present invention relates to a computer-implemented method for inferring MAPK-AP-1 cellular signaling pathway activity in a subject based on measured expression levels of three or more target genes of the MAPK-AP-1 cellular signaling pathway in a sample from the subject. The invention further relates to an apparatus, a non-transitory storage medium and a computer program for inferring MAPK-AP-1 cellular signaling pathway activity in a subject. The invention further relates to kits for measuring the expression levels of three or more target genes of the MAPK-AP-1 cellular signaling pathway in a sample from a subject, kits for inferring the activity of the MAPK-AP-1 cellular signaling pathway in a subject, and the use of these kits for performing the methods.)

1. A computer-implemented method performed by a digital processing apparatus for inferring MAPK-AP-1 cellular signaling pathway activity in a subject, wherein the inferring comprises:

receiving measured expression levels of three or more target genes of the MAPK-AP-1 cellular signaling pathway in a sample from the subject,

determining in the subject sample the level of activity of an AP-1Transcription Factor (TF) element, the AP-1TF element controlling transcription of the three or more AP-1 target genes, the determining based on evaluating a calibrated mathematical pathway model that correlates the expression levels of the three or more AP-1 target genes with the level of activity of the AP-1TF element, and

inferring activity of the MAPK-AP-1 cellular signaling pathway in the subject based on the determined level of activity of the AP-1TF element in the sample from the subject,

wherein the three or more AP-1 target genes are selected from: BCL2L11, CCND1, DDIT3, DNMT1, EGFR, ENPP2, EZR, FASLG, FIGF, GLRX, IL2, IVL, LOR, MMP1, MMP3, MMP9, SERPINE1, PLAU, PLAUR, PTGS2, SNCG, TIMP1, TP53 and VIM, preferably selected from: CCND1, EGFR, EZR, GLRX, MMP1, MMP3, PLAU, PLAUR, SERPINE1, SNCG and TIMP 1.

2. The method of claim 1, further comprising:

determining whether the MAPK-AP-1 cellular signaling pathway in the subject is operating abnormally based on a presumed activity of the MAPK-AP-1 cellular signaling pathway in the subject.

3. The method of claim 2, further comprising:

recommending to the subject a prescription for a drug to correct for abnormal operation of the MAPK-AP-1 cellular signaling pathway,

wherein the recommendation is made if the MAPK-AP-1 cell signaling pathway is determined to function abnormally in the subject based on the inferred MAPK-AP-1 cell signaling pathway activity.

4. The method of claim 2 or 3, wherein the abnormal operation of the MAPK-AP-1 cellular signaling pathway is operation of the MAPK-AP-1 cellular signaling pathway in a subject as a tumor promoter.

5. The method of any one of claims 1-4, wherein the method is used in at least one of the following activities:

a diagnosis based on the presumed activity of the MAPK-AP-1 cellular signaling pathway in the subject;

based on a prognosis of the presumed activity of the MAPK-AP-1 cellular signaling pathway in the subject;

a drug prescription based on the presumed activity of the MAPK-AP-1 cellular signaling pathway in the subject;

prediction of drug efficacy based on the presumed activity of the MAPK-AP-1 cellular signaling pathway in the subject;

predicting a side effect based on the presumed activity of the MAPK-AP-1 cellular signaling pathway in the subject;

monitoring the drug effect;

drug development;

measuring and developing;

research of a way;

staging of cancer;

recruitment of subjects in a clinical trial based on presumed MAPK-AP-1 cellular signaling pathway activity in the subjects;

selection of a subsequent test to be performed; and

with the selection of diagnostic tests.

6. The method of any one of claims 1-5, wherein the calibrated mathematical pathway model is a probabilistic model, preferably a Bayesian network model, based on conditional probabilities of correlating the activity level of the AP-1TF element with the expression levels of the three or more AP-1 target genes, or wherein the mathematical pathway model is based on one or more linear combinations of the expression levels of the three or more AP-1 target genes.

7. An apparatus for inferring MAPK-AP-1 cellular signaling pathway activity in a subject, comprising a digital processor configured to perform the method of any of claims 1-6.

8. A non-transitory storage medium for inferring MAPK-AP-1 cellular signaling pathway activity in a subject, storing instructions executable by a digital processing apparatus to perform the method of any of claims 1-6.

9. A computer program for inferring MAPK-AP-1 cellular signaling pathway activity in a subject, comprising program code means for causing a digital processing apparatus to carry out the method of any one of claims 1to 6 when said computer program is run on said digital processing apparatus.

10. A kit for measuring the expression levels of three or more target genes of the MAPK-AP-1 cellular signaling pathway in a sample of a subject, comprising:

polymerase chain reaction primers directed to the three or more AP-1 target genes,

probes for the three or more AP-1 target genes, and

the apparatus of claim 7, the non-transitory storage medium of claim 8 or the computer program of claim 9,

wherein the three or more AP-1 target genes are selected from: BCL2L11, CCND1, DDIT3, DNMT1, EGFR, ENPP2, EZR, FASLG, FIGF, GLRX, IL2, IVL, LOR, MMP1, MMP3, MMP9, SERPINE1, PLAU, PLAUR, PTGS2, SNCG, TIMP1, TP53 and VIM, preferably selected from: CCND1, EGFR, EZR, GLRX, MMP1, MMP3, PLAU, PLAUR, SERPINE1, SNCG and TIMP 1.

11. A kit for inferring MAPK-AP-1 cellular signaling pathway activity in a subject, comprising:

one or more components for determining the expression levels of three or more target genes of the MAPK-AP-1 cellular signaling pathway in a sample of a subject,

wherein said one or more components are preferably selected from: DNA array chips, oligonucleotide array chips, protein array chips, antibodies, a plurality of probes such as labeled probes, a set of RNA reverse transcriptase sequencing components, and/or RNA or DNA including cDNA amplification primers, and

the apparatus of claim 7, the non-transitory storage medium of claim 8 or the computer program of claim 9,

wherein the three or more AP-1 target genes are selected from: BCL2L11, CCND1, DDIT3, DNMT1, EGFR, ENPP2, EZR, FASLG, FIGF, GLRX, IL2, IVL, LOR, MMP1, MMP3, MMP9, SERPINE1, PLAU, PLAUR, PTGS2, SNCG, TIMP1, TP53 and VIM, preferably selected from: CCND1, EGFR, EZR, GLRX, MMP1, MMP3, PLAU, PLAUR, SERPINE1, SNCG and TIMP 1.

12. Use of a kit according to claims 10 and 11 for carrying out the method according to any one of claims 1to 6.

Technical Field

The present invention relates generally to bioinformatics, genomic processing, proteomic processing, and related fields. More particularly, the present invention relates to a computer-implemented method of inferring the activity of the MAPK-AP-1 cellular signaling pathway in a subject, performed by a digital processing apparatus, wherein the inference is based on measured expression levels of three or more target genes of the MAPK-AP-1 cellular signaling pathway in a sample of the subject. The invention also relates to an apparatus for inferring MAPK-AP-1 cellular signaling pathway activity in a subject, comprising a digital processor configured to perform the method, to a non-transitory storage medium for inferring MAPK-AP-1 cellular signaling pathway activity in a subject, storing instructions executable by a digital processing apparatus for performing the method, and to a computer program for inferring MAPK-AP-1 cellular signaling pathway activity in a subject, comprising program code means for causing a digital processing apparatus to perform the method when the computer program is run on the digital processing apparatus. The invention also relates to a kit for measuring the expression levels of three or more target genes of the MAPK-AP-1 cellular signaling pathway in a sample from a subject, to a kit for inferring the activity of the MAPK-AP-1 cellular signaling pathway in a subject, and to the use of said kit for carrying out said method.

Background

Genomic and proteomic analysis has been largely implemented in the medical field such as oncology and is expected to find clinical application, where various cancers are known to be associated with specific combinations of genomic mutations/variations and/or high or low expression levels of specific genes, which play a role in the growth and evolution of cancers such as cell proliferation and metastasis.

Aberrant MAPK pathway activity plays an important role in cancer and many other diseases. Activator protein 1(AP-1) is an inducible transcription factor that is strongly activated following MAPK stimulation. The AP-1transcription factor is a key target for MAPK signaling and regulates the expression of a variety of genes involved in proliferation, differentiation, and apoptosis, biological processes critical to cancer progression. AP-1 is primarily composed of Jun (e.g., c-Jun, JunB, and JunD) and/or Fos (e.g., c-Fos, FosB, Fra-1, and Fra-2) and/or ATF and/or JDP family members. In the nucleus, AP-1 binds to the promoter of the gene and induces a genetic program that promotes various cellular processes required for cancer progression (see also fig. 1).

With respect to signaling of MAPK in, for example, cancer, it is important to be able to detect aberrant MAPK-AP-1 signaling activity in order to be able to correctly select targeted drug therapy. anti-MAPK therapies are currently being used and new therapies are being developed (see Perego P. et al, "Modulation of cell sensitivity to inhibitors based on vital differentiation and survival pathways", Biochemical Pharmacology, Vol.80, No.10, pages1459to 1465). However, there are currently no clinical assays available for assessing the functional status with respect to MAPK-AP-1 cell signalling pathway activity, active (i.e. more likely to be tumor promoting than, for example, its inactive (passive) status. Accordingly, it would be desirable to improve the likelihood of identifying patients having a disease such as cancer, e.g., breast, lung, cervical, endometrial, ovarian, pancreatic or prostate cancer, or an immune disorder driven at least in part by aberrant MAPK-AP-1 cell signaling pathway activity, and thus likely to respond to inhibitors of MAPK-AP-1 cell signaling pathways.

Summary of The Invention

According to a broad aspect of the invention, the above problem is solved by a computer-implemented method performed by a digital processing apparatus for inferring activity of a MAPK-AP-1 cellular signaling pathway in a subject, wherein the inferring comprises:

receiving measured expression levels of three or more target genes, e.g., 3, 4, 5,6, 7, 8, 9, 10, 11 or more target genes, of the MAPK-AP-1 cellular signaling pathway in a sample of the subject,

determining in a sample of the subject the level of activity of an AP-1Transcription Factor (TF) element, the AP-1TF element controlling transcription of the three or more AP-1 target genes, the determining based on evaluating a calibrated mathematical model pathway that correlates the expression levels of the three or more AP-1 target genes with the level of activity of the AP-1TF element, and

inferring an activity of the MAPK-AP-1 cellular signaling pathway in the subject based on the determined level of activity of the AP-1TF element in the sample of the subject,

wherein the three or more AP-1 target genes are selected from: BCL2L11, CCND1, DDIT3, DNMT1, EGFR, ENPP2, EZR, FASLG, FIGF, GLRX, IL2, IVL, LOR, MMP1, MMP3, MMP9, SERPINE1, PLAU, PLAUR, PTGS2, SNCG, TIMP1, TP53 and VIM, preferably selected from: CCND1, EGFR, EZR, GLRX, MMP1, MMP3, PLAU, PLAUR, SERPINE1, SNCG and TIMP 1.

Herein, an "activity level" of a TF element refers to the level of activity of the TF element with respect to transcription of its target gene.

The present invention is based on the inventors' innovation that a suitable method of identifying a role occurring in the MAPK-AP-1 cellular signaling pathway can be based on measuring the signaling output of the MAPK-AP-1 cellular signaling pathway, which is, for example, transcription of a target gene, controlled by an AP-1Transcription Factor (TF) element controlled by the MAPK-AP-1 cellular signaling pathway. This innovation by the inventors assumes that the level of TF activity in the sample is in a quasi-stable state and can be detected by, for example, the expression value of the AP-1 target gene. The MAPK-AP-1 cellular signaling pathway targeted herein is known to control many functions of many cell types in humans, such as proliferation, differentiation and apoptosis. For pathological disorders, such as cancer (e.g., breast, cervical, lung, endometrial, ovarian, pancreatic or prostate cancer), aberrant MAPK-AP-1 cell signaling activity plays an important role and can be detected in the expression profile of target genes and thus exploited by calibrated mathematical pathway models.

The invention allows the activity of the MAPK-AP-1 cellular signaling pathway in a subject to be determined by: (i) determining the level of activity of an AP-1TF element in a sample from the subject, wherein the determining is based on evaluating a calibrated mathematical model that correlates the expression levels of three or more target genes of the MAPK-AP-1 cellular signaling pathway, the transcription of which is controlled by the AP-1TF element, with the level of activity of the AP-1TF element, and (ii) inferring the activity of the MAPK-AP-1 cellular signaling pathway in the subject based on the determined level of activity of the AP-1TF element in the sample from the subject. This preferably allows for an improved likelihood of identifying a patient having a disease, for example a cancer such as breast, cervical, lung, endometrial, ovarian, pancreatic or prostate cancer, which is driven at least in part by aberrant MAPK-AP-1 cell signalling pathway activity and therefore likely to respond to an inhibitor of the MAPK-AP-1 cell signalling pathway. In particular embodiments, treatment decisions may be based on specific MAPK-AP-1 cell signaling pathway activities. In a particular embodiment, the MAPK-AP-1 cell signaling status can be set as a cutoff value for the probability that the MAPK-AP-1 cell signaling pathway is active, e.g., 10:1, 5:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:5, or 1: 10.

In this context, the term "AP-1 transcription factor element" or "AP-1 TF element" or "TF element" is defined as a protein complex, it comprises at least one Jun (e.g., C-Jun, JunB and JunB) family member and/or Fos (e.g., C-Fos, FosB, Fra-1 and Fra-2) family member and/or ATF family member and/or JDP family member, forming, for example, Jun-Jun or Jun-Fos dimers capable of binding to a specific DNA sequence, preferably the response element 12-O-tetradecanoyl phorbol-13-ethyl ester (TPA), The Response Element (TRE) having the binding motif 5'-TGA G/C TCA-3' or the cyclic AMP response element (CRE) having the binding motif 5 '-CGTGA TCA-3', thereby controlling transcription of a target gene. Preferably, the term refers to a protein or protein complex transcription factor triggered by AP-1 inducing binding of ligands such as growth factors (e.g., EGF) and cytokines to their receptors or intermediate downstream signaling agents or triggered by the presence of an AP-1 activating mutation.

The calibrated mathematical pathway model may be a probabilistic model, preferably a Bayesian network model, based on conditional probabilities that correlate activity levels of AP-1TF elements with expression levels of the three or more AP-1 target genes, or the calibrated mathematical pathway model may be based on one or more linear combinations of expression levels of the three or more AP-1 target genes. In particular, the inference of MAPK-AP-1 cellular signaling pathway activity may be made as described in published International patent application WO2013/011479A2 ("Association of cellular signaling pathway activity using basic modulation of targeting expression") or as described in published International patent application WO2014/102668A2 ("association of cellular signaling pathway activity(s) of targeting gene expression"), which is incorporated herein in its entirety. Further details regarding the speculation of cell signaling pathway activity using mathematical modeling of target gene expression can be found in Verhaegh W.et al, "Selection of qualified pathway therapy through the use of knowledge-based regulatory models through pathways," Cancer Research, Vol.74, No.11,2014, pages 2936to 2945.

The term "subject" as used herein refers to any living organism. In some embodiments, the subject is an animal, preferably a mammal. In some embodiments, the subject is a human, preferably a medical subject. In other embodiments, the subject is a cell line.

The term "target gene" as used herein refers to a gene whose transcription is controlled directly or indirectly by an AP-1transcription factor element. The "target gene" can be a "direct target gene" and/or an "indirect target gene" (as described herein). In addition, "multiple target genes" can be "multiple direct target genes" and/or "multiple indirect target genes" (as described herein).

Particularly suitable AP-1 target genes are described in the following paragraphs and in the examples (see, e.g., tables 1 and 2 below).

Thus, according to a preferred embodiment, the AP-1 target gene is selected from the AP-1 target genes listed in Table 1 or Table 2 below.

The present inventors have found that a shorter list of AP-1 target genes is more likely to be responsible for determining MAPK-AP-1 cell signaling pathway activity.

Another aspect of the invention relates to a method (as described herein), further comprising:

determining whether the MAPK-AP-1 cellular signaling pathway in the subject is abnormally operating based on the presumed activity of the MAPK-AP-1 cellular signaling pathway in the subject.

The present invention also relates to a method (as described herein), further comprising:

recommending to the subject a prescription for a drug to correct abnormal operation of the MAPK-AP-1 cellular signaling pathway,

wherein the recommendation is made if the MAPK-AP-1 cell signaling pathway is determined to function abnormally in the subject based on the inferred MAPK-AP-1 cell signaling pathway activity.

The phrase "the cellular signaling pathway is abnormally operated" refers to a situation where "activity" of the pathway is not expected, wherein the term "activity" may refer to an activity of the transcription factor complex to drive expression of a target gene, i.e., the rate at which the target gene is transcribed. "normal" may refer to a tissue that is inactive in which it is expected to be inactive (inactive), but is active when it is expected to be active. Furthermore, a certain level of activity may be considered "normal", while any higher or lower activity may be considered "abnormal".

The invention also relates to a method (as described herein) wherein the abnormal operation of the MAPK-AP-1 cellular signaling pathway is an operation in which the MAPK-AP-1 cellular signaling pathway operates as a tumor promoter (promoter) in a subject.

The sample used in the present invention may be an extracted sample, i.e. a sample that has been extracted from a subject. Examples of samples include, but are not limited to, tissues, cells, blood, and/or bodily fluids of a subject. If the subject is a medical subject suffering from or likely to suffer from cancer or other diseases such as autoimmune diseases, it may be, for example, a sample obtained from: cancer lesions, lesions suspected of having cancer, metastatic tumors, body cavities (e.g. thoracic or abdominal or bladder cavities) where body fluids contaminated with cancer cells are present or other body fluids containing cancer cells, or samples from affected tissues or cells etc. in case of patients with other diseases, are preferably obtained by biopsy procedures or other sample extraction procedures. The cells from which the sample is taken may also be tumor cells of a hematological malignancy (e.g., leukemia or lymphoma). In some cases, the cell sample may also be circulating tumor cells, i.e., tumor cells that have entered the bloodstream and may be extracted using a suitable separation technique, such as apheresis or conventional venous blood draw. In addition to blood, the bodily fluid from which the sample is extracted may be urine, gastrointestinal contents or exudates. The term "sample" as used herein also covers the situation where e.g. tissue and/or cells and/or bodily fluids of a subject have been taken from the subject and e.g. placed on a microscope slide, and a portion of the sample has been extracted for the purpose of the claimed method, e.g. by Laser Capture Microdissection (LCM), or scraping of cells of interest from a slide, or by fluorescence activated cell sorting techniques. In addition, the term "sample" as used herein also covers the situation where e.g. tissue and/or cells and/or body fluids of a subject have been taken from the subject and placed on a microscope slide on which the method of claim is performed.

According to another disclosed aspect, an apparatus for inferring MAPK-AP-1 cellular signaling pathway activity in a subject includes a digital processor configured to perform the inventive methods described herein.

According to another disclosed aspect, a non-transitory storage medium for inferring MAPK-AP-1 cellular signaling pathway activity in a subject stores instructions executable by a digital processing apparatus to perform the inventive methods described herein. The non-transitory storage medium may be a computer-readable storage medium, such as a hard disk drive or other magnetic storage medium, an optical disk or other optical storage medium, Random Access Memory (RAM), Read Only Memory (ROM), flash memory or other electronic storage medium, a network server, or the like. The digital processing device may be a handheld device (e.g., a personal data assistant or smart phone), a notebook computer, a desktop computer, a tablet computer or device, a remote web server, or the like.

According to another disclosed aspect, a computer program for inferring MAPK-AP-1 cellular signaling pathway activity in a subject comprises program code means for causing a digital processing apparatus to carry out the inventive methods described herein when said computer program is run on said digital processing apparatus. The digital processing device may be a handheld device (e.g., a personal data assistant or smart phone), a notebook computer, a desktop computer, a tablet computer or device, a remote web server, or the like.

According to another disclosed aspect, a kit for measuring the expression level of three or more, e.g., 3, 4, 5,6, 7, 8, 9, 10, 11 or more target genes of the MAPK-AP-1 cellular signaling pathway in a sample of a subject comprises:

one or more components for determining the expression levels of the three or more AP-1 target genes in a sample from a subject,

wherein the three or more AP-1 target genes are selected from: BCL2L11, CCND1, DDIT3, DNMT1, EGFR, ENPP2, EZR, FASLG, FIGF, GLRX, IL2, IVL, LOR, MMP1, MMP3, MMP9, SERPINE1, PLAU, PLAUR, PTGS2, SNCG, TIMP1, TP53 and VIM, preferably selected from: CCND1, EGFR, EZR, GLRX, MMP1, MMP3, PLAU, PLAUR, SERPINE1, SNCG and TIMP 1.

The one or more components or means for measuring the expression levels of the three or more AP-1 target genes may be selected from: DNA array chips, oligonucleotide array chips, protein array chips, antibodies, a plurality of probes such as labeled probes, a set of RNA reverse transcriptase sequencing components and/or RNA or DNA (including cDNA) amplification primers. In one embodiment, the kit comprises a set of labeled probes directed to a portion of the mRNA or cDNA sequence of three or more AP-1 target genes described herein. In one embodiment, the kit comprises a set of primers and probes directed to a portion of the mRNA or cDNA sequence of the three or more AP-1 target genes. In one embodiment, the labeled probe is contained in a standardized 96-well plate. In one embodiment, the kit further comprises primers or probes directed against a set of reference genes. Such a reference gene may be, for example, a constitutively expressed gene for normalizing or normalizing the expression level of a target gene described herein.

In one embodiment, the kit for measuring the expression level of three or more, e.g., 3, 4, 5,6, 7, 8, 9, 10, 11 or more target genes of the MAPK-AP-1 cellular signaling pathway in a sample of a subject comprises:

polymerase chain reaction primers directed to the three or more AP-1 target genes,

probes for the three or more AP-1 target genes, and

wherein the three or more AP-1 target genes are selected from: BCL2L11, CCND1, DDIT3, DNMT1, EGFR, ENPP2, EZR, FASLG, FIGF, GLRX, IL2, IVL, LOR, MMP1, MMP3, MMP9, SERPINE1, PLAU, PLAUR, PTGS2, SNCG, TIMP1, TP53 and VIM, preferably selected from: CCND1, EGFR, EZR, GLRX, MMP1, MMP3, PLAU, PLAUR, SERPINE1, SNCG and TIMP 1.

According to another disclosed aspect, a kit for predicting the activity of the MAPK-AP-1 cellular signaling pathway in a subject comprises:

the kits of the invention described herein, and

the inventive apparatus described herein, the inventive non-transitory storage medium described herein or the inventive computer program described herein.

According to another disclosed aspect, the kits of the invention described herein are used to perform the methods of the invention described herein.

The invention described herein may also, for example, be advantageously used in at least one of the following activities:

a diagnosis based on the presumed activity of the MAPK-AP-1 cellular signaling pathway in the subject;

based on a prognosis of the presumed activity of the MAPK-AP-1 cellular signaling pathway in the subject;

a drug prescription based on the presumed activity of the MAPK-AP-1 cellular signaling pathway in the subject;

prediction of drug efficacy based on the presumed activity of the MAPK-AP-1 cellular signaling pathway in the subject;

predicting a side effect based on the presumed activity of the MAPK-AP-1 cellular signaling pathway in the subject;

monitoring the drug effect;

drug development;

measuring and developing;

research of a way;

staging of cancer;

recruitment of subjects based on presumed MAPK-AP-1 cellular signaling pathway activity in the subjects;

selection of a subsequent test to be performed; and

with the selection of diagnostic tests.

Further advantages will become apparent to those of ordinary skill in the art upon reading and understanding the drawings, the following description and especially the detailed embodiments provided below.

It shall be understood that the method of claim 1, the device of claim 7, the non-transitory storage medium of claim 8, the computer program of claim 9, the kit of claims 10-12 and the use of the kit of claim 13 have similar and/or identical preferred embodiments, in particular as defined in the dependent claims.

It shall be understood that preferred embodiments of the invention can also be any combination of the dependent claims or the above embodiments with the respective independent claims.

These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

Brief Description of Drawings

FIG. 1 schematically and exemplarily shows a MAPK-AP-1 cell signaling pathway. Activin 1(AP-1) is an inducible transcription factor that is strongly activated following MAPK stimulation. The AP-1transcription factor is a key target for MAPK-AP-1 signaling, regulating the expression of a variety of genes involved in proliferation, differentiation and apoptosis, biological processes critical to cancer progression. AP-1 is primarily composed of Jun (e.g., c-Jun, JunB, and JunD) and/or Fos (e.g., c-Fos, FosB, Fra-1, and Fra-2) and/or ATF and/or JDP family members. In the nucleus, AP-1 binds to the promoter of the gene and induces a genetic program that promotes a variety of cellular processes required for cancer progression.

FIG. 2 schematically and exemplarily shows a mathematical model, herein a Bayesian network model, for modeling transcription programs of the MAPK-AP-1 cell signaling pathway.

FIG. 3 shows a flow chart illustrating a process for inferring MAPK-AP-1 cellular signaling pathway activity in a subject based on measured expression levels of target genes of the MAPK-AP-1 cellular signaling pathway in a sample from the subject.

FIG. 4 shows a flow chart illustrating a process for obtaining a calibrated mathematical pathway model as described herein.

FIG. 5 shows a flow chart illustrating a process for determining the level of activity of an AP-1Transcription Factor (TF) element in a subject sample as described herein.

Figure 6 shows a flow chart exemplarily illustrating a process for inferring MAPK-AP-1 cellular signaling pathway activity in a subject using discrete observables.

Figure 7 shows a flow chart illustrating a process for using continuous observables to infer MAPK-AP-1 cellular signaling pathway activity in a subject.

FIG. 8 shows a flow chart illustrating a process for determining Cq values from RT-qPCR analysis of target genes of the MAPK-AP-1 cell signaling pathway.

Figure 9 shows MAPK-AP-1 cell signaling pathway activity predictions for 18 samples from GSE28878 using the target gene evidence summary list (24 target gene list) of table 1 trained on an exemplary bayesian network model.

Figure 10 shows MAPK-AP-1 cell signaling pathway activity prediction for 12 samples from GSE45417 using the target gene evidence summary list of table 1 (24 target gene list) trained exemplary bayesian network model.

Figure 11 shows MAPK-AP-1 cell signaling pathway activity prediction for 18 samples from GSE66853 using the target gene evidence summary list of table 1 (24 target gene list) trained exemplary bayesian network model.

Figure 12 shows MAPK-AP-1 cell signaling pathway activity prediction for 2 samples from E-MEXP-2213 using the target gene evidence summary list of table 1 (24 target gene list) trained exemplary bayesian network model.

Figure 13 shows MAPK-AP-1 cell signaling pathway activity predictions for 25 samples from GSE2677, GSE2842 and GSE39338 using the target gene evidence summary list of table 1 (24 target gene list) trained exemplary bayesian network model.

Figure 14 shows MAPK-AP-1 cell signaling pathway activity prediction for 15 samples from GSE40117 using the 11 target gene enrollment list (shortlist) from table 2 trained an exemplary bayesian network model.

Figure 15 shows MAPK-AP-1 cell signaling pathway activity prediction for 18 samples from GSE58235 using the 11 target gene enrollment list from table 2 trained exemplary bayesian network model.

Figure 16 shows MAPK-AP-1 cell signaling pathway activity prediction for 12 samples from E-MEXP-2573 using the 11 target gene enrollment list from table 2 trained exemplary bayesian network models.

Figure 17 shows MAPK-AP-1 cell signaling pathway activity prediction for 56 samples from GSE21618 using the 11 target gene enrollment list from table 2 trained an exemplary bayesian network model.

Figure 18 shows the results of further validation of breast cancer subgroups in samples from GSE6532, GSE9195, GSE12276, GSE20685, GSE21653, GSE58812, GSE66986, GSE102484, and E-MTAB-365 using the 11 target gene enrollment list from table 2 trained for the exemplary bayesian network model.

FIG. 19 shows a Kaplan-Meier curve for breast cancer patients with high AP-1 activity (tested with a trained exemplary Bayesian network model using the summary list of target gene evidence (list of 24 target genes) in Table 1).

Fig. 20 shows the results of further validation of breast cancer subgroups of samples from GSE5060, GSE10006, GSE10245, GSE13933, GSE19667, GSE28582, GSE30219, GSE33532, GSE43346, and GSE50081 using the trained exemplary bayesian network model using the target gene evidence summary list (24 target gene list) from table 1.

Fig. 21 shows the correlation between the exemplary bayesian network model trained on 5307 samples from the common dataset using the target gene evidence summary list from table 1 (24 target gene list) and the 11 target gene enrollment list from table 2, respectively.

Detailed description of the embodiments

The following examples illustrate only particularly preferred methods and selected aspects associated therewith. The teachings provided herein can be used to construct assays and/or kits, for example, to detect, predict, and/or diagnose abnormal activity of the MAPK-AP-1 cellular signaling pathway. In addition, when using the methods described herein, it may be advantageous to prescribe drugs, to perform predictions of drug response and monitoring of drug efficacy (and/or side effects), to predict and monitor drug resistance, for example to select subsequent tests to be performed (e.g., concomitant diagnostic tests). The following examples should not be construed as limiting the scope of the invention.