Method for determining hippo pathway activity

Abstract

The invention describes a method to infer the Hippo cellular signaling pathway activity in a sample based on assessing the expression levels of six or more predefined target genes. The invention further describes the use of the method in prognostic and diagnostic applications and use of the method to determine or predict therapy resistance.

Description

[0001] Title: Method for determining Hippo pathway activity

[0002] Field of the invention

[0003] The present invention generally relates to the field of bioinformatics, genomic processing, proteomic processing, and related arts. More particularly, the present invention relates to a computer-implemented method for inferring activity of a Hippo cellular signaling pathway in a sample performed by a digital processing device, wherein the inferring is based on gene expression levels of six or more target genes of the Hippo cellular signaling pathway measured in a sample of the subject. The invention further relates to methods of predicting an outcome of a subject based on the inferred Hippo pathway activity as determined in a sample obtained from the subject. For example the method may provide an outcome for a subject with cancer. In particular the method may be useful in determining resistance of a tumor with a KRAS mutation to KRAS inhibitors.

[0004] Background of the invention

[0005] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0006] Genomic and proteomic analyses have substantial realized and potential promise for clinical application in medical fields such as oncology and immunology, where various cancers are known to be associated with specific combinations of genomic mutations / variations and / or high or low gene expression levels for specific genes, which play a role in growth and evolution of cancer, e.g., cell proliferation and metastasis.

[0007] The Hippo signaling pathway is a signaling pathway that controls organ size in animals through the regulation of cell proliferation and apoptosis. In mammals the MST1 / 2 kinases phosphorylate the protein kinases LATS1 / 2 which become active upon phosphorylation. The LATS1 / 2 kinases in turn phosphorylate YAP and TAZ, which are transcription co-activators. YAP and TAZ when not phosphorylated can bind to TEAD (TEAD1 / 2 / 3 / 4) and translocate to the nucleus to initiate transcription (or repression) of target genes. Dysregulation of the Hippo pathway may be involved in a variety of diseases, including cancer, eye diseases, cardiac diseases, pulmonary diseases, renal diseases, hepatic diseases, and immune dysfunction. Therefore, therapeutic strategies that target dysregulated Hippo components are being investigated as promising tools for the treatment of a wide spectrum of diseases. In addition, measuring and monitoring Hippo pathway activity may provide valuable tools for prognostics, therapy prediction or evaluating therapy success.

[0008] For the development, testing and prescription of therapies intervening with the Hippo pathway reliable tests are needed to asses Hippo pathway activity. Presently no accurate tests are known.

[0009] Therefore there is a continuing need for improved methods for assessing Hippo pathway activity in a sample. Further there is need for methods for prognosis and therapy recommendation based on Hippo pathway activity. This need is met by the methods and products as defined in the appended claims.

[0010] Figures

[0011] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0012] Fig. 1 depicts the assessed activity of the MAPK, PI3K, NFkB, WNT and Hippo cellular signaling pathways in NCI-H358 (top graph) or NCI-H23 cells mock treated or treated with sotorasib (either sensitive or resistant cells).

[0013] Fig. 2 depicts the assessed activity of the MAPK, PI3K, NFkB, WNT and Hippo cellular signaling pathways in OVCAR8 (top graph) or HCC1576 cells mock treated or treated with GNE-7883 or putative YAP inhibitors compounds 2-4. of the invention

[0014] As embodied and broadly described herein, the present invention is directed to the surprising finding that the Hippo cellular signaling pathway activity can be accurately be determined by assessing the gene expression levels of the herein described target genes. In addition the invention is directed to the finding that methods to determine Hippo cellular signaling pathway as broadly described herein may be used to predict or to determine resistance of a tumor to treatment with a KRAS inhibitor. Therefore, in a first aspect the invention describes a method for inferring the activity of the Hippo cellular signaling pathway in a sample, the method comprising receiving the gene expression levels of six or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2, the gene expression levels of six or more Hippo target genes being determined in a sample; and inferring the Hippo cellular signaling pathway activity based on the six or more gene expression levels.

[0015] In a second aspect the invention describes a method for determining or predicting resistance of a tumor to a KRAS inhibitor, the method comprising receiving the gene expression levels of six or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2, the gene expression levels of six or more Hippo target genes being determined in a sample obtained from the tumor; inferring the Hippo cellular signaling pathway activity based on the six or more gene expression levels, and predicting or determining the tumor to be or become resistant to KRAS inhibitors if the inferred Hippo cellular signaling pathway activity is low.

[0016] In a third aspect the invention describes a KRAS inhibitor for use in the treatment, prevention or amelioration of cancer in a subject, the use comprising receiving the gene expression levels of six or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2, the gene expression levels of six or more Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the six or more gene expression levels, and administering the KRAS inhibitor when the Hippo pathway is active.

[0017] In a fourth aspect the invention describes a YAP / TAZ inhibitor for use in the treatment, prevention or amelioration of cancer in a subject, the use comprising receiving the gene expression levels of six or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2, the gene expression levels of six or more Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the six or more gene expression levels, and administering the YAP / TAZ inhibitor when the Hippo pathway is inactive.

[0018] In a fifth aspect the invention describes a Hippo activator for use in the treatment, prevention or amelioration of cancer in a subject, the use comprising receiving the gene expression levels of six or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1, WWC1 , and NLIAK2, the gene expression levels of six or more Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the six or more gene expression levels, and administering the Hippo activator when the Hippo pathway is inactive.

[0019] In a sixth aspect the invention describes a method of treating, preventing or ameliorating cancer in a subject in need thereof, the method comprising receiving the gene expression levels of six or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2, the gene expression levels of six or more Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the six or more gene expression levels, and if the Hippo pathway is active administering a KRAS inhibitor to the subject; or if the Hippo pathway is inactive administering a Hippo activator optionally combined with a KRAS inhibitor, a YAP / TAZ inhibitor optionally combined with a KRAS inhibitor or an alternative therapy selected from chemotherapy, surgery, immunotherapy, radiation therapy, or a combination thereof.

[0020] In a seventh aspect the invention describes a kit of parts comprising polymerase chain reaction primers and optionally probes for specifically determining the gene expression levels of six or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2.

[0021] In an eight aspect the invention describes the use of the kit according to the seventh aspect of the invention in inferring the Hippo cellular signaling pathway activity, the use comprising determining the gene expression levels of six or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2 using the polymerase chain reaction primers and optionally probes provided in the kit and inferring the Hippo cellular signaling pathway based on the gene expression levels of the six or more Hippo target genes.

[0022] Definitions

[0023] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0024] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. For purposes of the present invention, the following terms are defined below.

[0025] As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, “a method for providing a cell” includes the providing of a plurality of cells (e.g. 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more cells). For example, “a first target gene” includes providing a plurality of such “first target genes”.

[0026] The terms “about” and “approximately”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1% and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. As used herein, the term “and / or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0027] As used herein, the term "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... , etc. As used herein, the term "at most" a particular value means that particular value or less. For example, "at most 5" is understood to be the same as "5 or less" i.e., 5, 4, 3, ... .-10, -11 , etc.

[0028] As used herein, the word “comprise” or variations thereof such as “comprises” or “comprising” will be understood to include a stated element, integer or step, or group of elements, integers or steps, but not to exclude any other element, integer or steps, or groups of elements, integers or steps. The verb “comprising” includes the verbs “essentially consisting of” and “consisting of”.

[0029] As used herein, “conventional techniques” or “methods known to the skilled person” refer to a situation wherein the methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology and related fields are well-known to those of skill in the art and are discussed, in various handbooks and literature references.

[0030] As used herein, "exemplary" or “for example” means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations, including those disclosed herein.

[0031] When used herein the term “sample” refers to a source tumor cells. A sample is preferably obtained from a human or non-human animal test subject, but may also include in vitro samples such as but not limited to a cell culture medium or culture supernatant. When obtained from a test subject the sample may refer to a tumor biopsy, a tissue sample, a blood sample, a plasma sample, a saliva sample, a stool sample, a urine sample, a cerebrospinal fluid sample, or a tissue derived fluid sample. Preferably the sample is a tumor biopsy or a tissue sample.

[0032] When used herein the terms “Hippo pathway”, “Hippo signaling pathway” and “hippo cellular signaling pathway” are used interchangeably and intend to refer to the signaling pathway that controls the phosphorylation of the YAP and TAZ co-regulators of transcription. Thus the Hippo pathway may include components such as but not limited to MST1 , MST2, LATS1 , and LATS2. Phosphorylation of the YPA and / or TAZ transcription co-regulators results in cytoplasmic retention or degradation of these proteins. In unphosphorylated state YAP and / or TAZ can translocate to the nucleus and initiate transcription with a cofactor such as TEAD1 , TEAD2, TEAD3, or TEAD4. Thus an active Hippo pathway refers to the situation where YAP and / or TAZ mediated transcription of target genes is low and an inactive Hippo pathway refers to the situation where YAP and / or TAZ mediated transcription of target genes is high. Thus an active Hippo pathway also refers to the situation where YAP and / or TAZ are predominantly phosphorylated and located in the cytoplasm where an inactive Hippo pathway refers to the situation where a substantial part of the YAP and / or TAZ proteins in the cells are phosphorylated and located in the nucleus, allowing transcription of YAP / TAZ target genes.

[0033] When used herein a Hippo target gene refers to a target gene is controlled by the Hippo pathway. Therefore a Hippo target gene is typically under the control of the YAP and / or TAZ transcription complex, which is negatively regulated by the Hippo pathway. Thus genes whose transcription is upregulated or initiated by the YAP and / or TAZ transcription complex are negatively regulated by Hippo (meaning their expression is suppressed) and vice versa genes whose transcription is downregulated or inhibited by the YAP and / or TAZ transcription complex are positively regulated by Hippo (meaning their expression is increased or initiated). Many Hippo target genes have been described in the literature. When referring to Hippo target genes according to the invention herein is meant specifically the target genes NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2, which have specifically been validated and curated for use in a Hippo pathway activity model.

[0034] Detailed description of the invention

[0035] The invention is defined herein, and in particular in the accompanying claims. Subjectmatter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[0036] It is contemplated that any method, use, or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[0037] Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions, and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.

[0038] All cited references, both patent publications and journal articles, are hereby incorporated by reference in their entirety for all uses and purposes.

[0039] As embodied and broadly described herein, the present invention is directed to the surprising finding that the Hippo cellular signaling pathway can be accurately determined based on the gene expression levels of three or more, preferably six or more target genes defined herein. The invention further describes several implementations of this method in prognosis and therapy response prediction as detailed below.

[0040] The present invention is based on the innovation of the inventors that a suitable way of identifying effects occurring in the Hippo cellular signaling pathway can be based on a measurement of the signaling output of the Hippo cellular signaling pathway, which is - amongst others - the transcription of the target genes, which is controlled by a Hippo transcription factor (TF) element that is controlled by the Hippo cellular signaling pathway. This innovation by the inventors assumes that the TF activity level is at a quasi-steady state in the sample, which can be detected by means of - amongst others - the gene expression values of the Hippo target genes. The Hippo cellular signaling pathway targeted herein is known to be involved the pathological process of eye diseases, cardiac diseases, pulmonary diseases, renal diseases, hepatic diseases, immune system-related diseases such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. The Hippo pathway is further closely related to organ development, stem cell biology, regeneration, and tumor biology, and known to affect cell differentiation by participating in extracellular and intracellular physiological signal reactions, sensing cell environment, and coordinating cell reactions.

[0041] The Hippo cellular signaling pathway can be regulated by upstream factors such as cell polarity, mechanical cues such as ECM density, cell geometry, liquid flow force, and cytoskeleton tension, or factors such as cell density, soluble factors (e.g. GPCR signaling), stress signals, and cell growth, proliferation and differentiation. Most notably an active Hippo pathway inhibits proliferation.

[0042] Dysregulation of the Hippo pathway can cause a variety of diseases, including cancer, eye diseases, cardiac diseases, pulmonary diseases, renal diseases, hepatic diseases, and immune dysfunction (see e.g. Fu et al. Signal Transduction and Targeted Therapy (2022) 7:376 for a review). Overwhelming evidence suggested that the Hippo pathway is one of the most frequently dysregulated pathways in human cancer; YAP / TAZ have been identified as oncoproteins, while MST1 / 2 and LATS1 / 2 have been identified as tumor suppressors. Cancers such as uveal melanoma, mesothelioma, ependymoma, and NF2-related schwannomas have all been shown to be related to Hippo pathway dysregulation. Generally, the Hippo pathway can affect human cancer in three ways: tumor initiation and progression, tumor metastasis, and tumor drug resistance. The Hippo pathway is an essential survival-associated signaling pathway, inactivation of this pathway could increase cell proliferation and decrease apoptosis, contributing to tumor initiation and progression. In addition Cancer stem cells (CSCs), which are a subpopulation of cancer cells, play an important role in tumor initiation and progression. The Hippo pathway component TAZ potentiates CSCs and depletion of TAZ significantly decreases the tumor-seeding ability of CSCs. Activation of the Hippo pathway or inhibition of YAP have also been shown to reduce metastasis in in vivo animal models. Finally, the most important clinical implication is the involvement of the Hippo pathway in targeted therapy resistance. BRAF inhibitors can be used to treat BRAF-mutant melanoma, however, efficacy is limited because of drug resistance. Upregulation of MOB3B and activation of the Hippo pathway contribute to vemurafenib resistance. In addition, NF2 is involved in vemurafenib resistance. The Hippo pathway likely plays an essential role in MEK inhibitor resistance (Fu et al. Signal Transduction and Targeted Therapy (2022) 7:376). In addition the Hippo pathway is linked to KRAS inhibitor resistance (Hagenbeek, et al. An allosteric pan-TEAD inhibitor blocks oncogenic YAP / TAZ signaling and overcomes KRAS G12C inhibitor resistance. Nat Cancer 4, 812-828 (2023)). Thus assessing the Hippo pathway in a sample may be useful for prognostic and diagnostic applications, companion diagnostics, monitoring of treatment effect, as well as screening therapeutic compounds.

[0043] Therefore, in a first aspect the invention describes a method for inferring the activity of the Hippo cellular signaling pathway in a sample, the method comprising receiving the gene expression levels of three or more, preferably six or more, for example three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two or even all twenty three, Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2, the gene expression levels of three or more, preferably six or more, Hippo target genes being determined in a sample; and inferring the Hippo cellular signaling pathway activity based on the three or more, preferably six or more, gene expression levels.

[0044] In a more preferred embodiment the three or more, preferably six or more, gene expression levels comprise or consist of three or more, preferably six or more, for example six, seven, eight, nine, or all ten, Hippo target gene expression levels selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, WWC1.

[0045] It is further envisioned that the Methods and uses described herein are based on fewer gene expression levels, for example three, four or five gene expression levels selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2, or selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, WWC1.

[0046] The present invention makes it possible to determine the activity of the Hippo cellular signaling pathway in a sample by determining or receiving the gene expression levels of three or more, preferably six or more, Hippo target genes as defined herein, and inferring the Hippo cellular signaling pathway activity based on the three or more, preferably six or more gene expression levels. An exemplary basic pathway model can be constructed by simply adding up the gene expression levels of the three or more, preferably six or more, target genes to arrive at a numerical value representing a pathway activity. Alternatively each gene expression level of each of the three or more, preferably six or more, target genes is individually normalized by multiplication by a predetermined constant prior to adding up the values to arrive at a numerical value representing the Hippo cellular signaling pathway activity.

[0047] This preferably allows improving the possibilities of characterizing patients that have a disease, such as eye diseases, cardiac diseases, pulmonary diseases, renal diseases, hepatic diseases, an immune system-related diseases such as rheumatoid arthritis, inflammatory bowel disease and psoriasis, or cancer, which is at least partially driven by an abnormal activity of the Hippo cellular signaling pathway, and that are therefore likely to respond to activators or inhibitors of the Hippo cellular signaling pathway. Thus, in cases where the Hippo cellular signaling pathway is acting as a protective (e.g. as tumor suppressive) pathway, an abnormally low activity of the pathway may preferably be identified using the methods of the present invention and a treatment that increases the activity of the Hippo cellular signaling pathway, for instance, by providing [...] to the patient, may be given to the patient. In particular embodiments, treatment determination can be based on a specific Hippo cellular signaling pathway activity. In a particular embodiment, the Hippo cellular signaling status can be set at a cutoff value of odds of the Hippo cellular signaling pathway being active of, for example, 10:1 , 5: 1 , 4:1 , 2:1 , 1 :1 , 1 :2, 1 :4, 1 :5, or 1 : 10.

[0048] The term “NPPB” refers to the human natriuretic peptide precursor B gene (Ensembl: ENSG00000120937), for example, to the sequence as defined in NCBI Reference Sequence NM_002521 .3, specifically, to the nucleotide sequence as set forth in SEQ ID NO:1 , which corresponds to the sequence of the above indicated NCBI Reference Sequences of the NPPB transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:2, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_002512.1 encoding the NPPB polypeptide.

[0049] The term “NPPB” also comprises nucleotide sequences showing a high degree of homology to NPPB, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:1 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:2 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:2 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 1.

[0050] The term “ANKRD1” refers to the human ankyrin repeat domain 1 gene (Ensembl: ENSG00000148677), for example, to the sequence as defined in NCBI Reference Sequence NM_014391.3, specifically, to the nucleotide sequence as set forth in SEQ ID NO:3, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the ANKRD1 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:4, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_055206.2 encoding the ANKRD1 polypeptide.

[0051] The term “ANKRD1” also comprises nucleotide sequences showing a high degree of homology to ANKRD1 , e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:3 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:4 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:4 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:3.

[0052] The term “IGFBP3” refers to the human insulin like growth factor binding protein 3 gene (Ensembl: ENSG00000146674), for example, to the sequence as defined in NCBI Reference Sequence NM_001013398.2, specifically, to the nucleotide sequence as set forth in SEQ I D NO:5, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the IGFBP3 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:6, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_001013416.1 encoding the IGFBP3 polypeptide.

[0053] The term “IGFBP3” also comprises nucleotide sequences showing a high degree of homology to IGFBP3, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:5 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:6 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:6 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:5.

[0054] The term “CCN2” refers to the human cellular communication network factor 2 gene (Ensembl: ENSG00000118523), for example, to the sequence as defined in NCBI Reference Sequence NM_001901.4, specifically, to the nucleotide sequence as set forth in SEQ ID NO:7, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the CCN2 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:8, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_001892.2 encoding the CCN2polypeptide.

[0055] The term “CCN2” also comprises nucleotide sequences showing a high degree of homology to CCN2, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:7 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:8 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:8 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:7.

[0056] The term “F3” refers to the human tissue factor gene (Ensembl: ENSG00000117525), for example, to the sequence as defined in NCBI Reference Sequence NM_001993.5, specifically, to the nucleotide sequence as set forth in SEQ ID NO:9, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the F3 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:10, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_001984.1 encoding the F3 polypeptide.

[0057] The term “F3” also comprises nucleotide sequences showing a high degree of homology to F3, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:9 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NQ: 10 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NQ: 10 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:9.

[0058] The term “KISS1” refers to the human kisspeptin gene (Ensembl: ENSG00000170498 ), for example, to the sequence as defined in NCBI Reference Sequence NM_002256.4, specifically, to the nucleotide sequence as set forth in SEQ ID NO:11 , which corresponds to the sequence of the above indicated NCBI Reference Sequences of the KISS1 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO: 12, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_002247.3 encoding the KISS1 polypeptide.

[0059] The term “KISS1” also comprises nucleotide sequences showing a high degree of homology to KISS1 , e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 11 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 12 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 12 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:11. The term “SNAPC1” refers to the human snRNA-activating protein complex subunit 1 gene (Ensembl: ENSG00000023608), for example, to the sequence as defined in NCBI Reference Sequence NM_003082.4, specifically, to the nucleotide sequence as set forth in SEQ ID NO: 13, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the SNAPC1 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO: 14, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_003073.1 encoding the SNAPC1 polypeptide.

[0060] The term “SNAPC1” also comprises nucleotide sequences showing a high degree of homology to SNAPC1 , e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 13 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 14 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 14 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 13.

[0061] The term “CCN1” refers to the human cellular communication network factor 1 gene (Ensembl: ENSG00000142871), for example, to the sequence as defined in NCBI Reference Sequence NM_001554.5, specifically, to the nucleotide sequence as set forth in SEQ ID NO: 15, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the CCN1 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:16, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_001545.2 encoding the CCN1 polypeptide.

[0062] The term “CCN1” also comprises nucleotide sequences showing a high degree of homology to CCNI , e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 15 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:16 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 16 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:15.

[0063] The term “CCND1” refers to the human cyclin D1 gene (Ensembl: ENSG00000110092), for example, to the sequence as defined in NCBI Reference Sequence NM_053056.3, specifically, to the nucleotide sequence as set forth in SEQ ID NO: 17, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the CCND1 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO: 18, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_444284.1 encoding the CCND1 polypeptide.

[0064] The term “CCND1” also comprises nucleotide sequences showing a high degree of homology to CCND1 , e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 17 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 18 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 18 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 17.

[0065] The term “AMOTL2” refers to the human Angiomotin-like protein 2 gene (Ensembl: ENSG00000114019), for example, to the sequence as defined in NCBI Reference Sequence NM_001278683.1 , specifically, to the nucleotide sequence as set forth in SEQ ID NO:19, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the AMOTL2 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NQ:20, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_001265612.1 encoding the AMOTL2 polypeptide. The term “AMOTL2” also comprises nucleotide sequences showing a high degree of homology to AMOTL2, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 19 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:20 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NQ:20 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:19.

[0066] The term “ITGB2” refers to the human Integrin beta chain-2 (CD18) gene (Ensembl: ENSG00000160255), for example, to the sequence as defined in NCBI Reference Sequence NM_000211.5, specifically, to the nucleotide sequence as set forth in SEQ ID NO:21 , which corresponds to the sequence of the above indicated NCBI Reference Sequences of the ITGB2 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:22, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_000202.3 encoding the ITGB2 polypeptide.

[0067] The term “ITGB2” also comprises nucleotide sequences showing a high degree of homology to ITGB2, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:21 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:22 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:22 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:21.

[0068] The term “SKP2” refers to the human S-phase kinase-associated protein 2 gene (Ensembl: ENSG00000145604), for example, to the sequence as defined in NCBI Reference Sequence NM_001243120.2, specifically, to the nucleotide sequence as set forth in SEQ ID NO:23, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the SKP2 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:24, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_001230049.1 encoding the SKP2 polypeptide.

[0069] The term “SKP2” also comprises nucleotide sequences showing a high degree of homology to SKP2, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:23 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:24 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:24 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:23.

[0070] The term “DKK1” refers to the human Dickkopf-related protein 1 gene (Ensembl: ENSG00000107984), for example, to the sequence as defined in NCBI Reference Sequence NM_012242.4, specifically, to the nucleotide sequence as set forth in SEQ ID NO:25, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the DKK1 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:26, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_036374.1 encoding the DKK1 polypeptide.

[0071] The term “DKK1” also comprises nucleotide sequences showing a high degree of homology to DKK1 , e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:25 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:26 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:26 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:25.

[0072] The term “AXL” refers to the human Tyrosine-protein kinase receptor UFO gene (Ensembl: ENSG00000167601), for example, to the sequence as defined in NCBI Reference Sequence NM_001278599.2, specifically, to the nucleotide sequence as set forth in SEQ ID NO:27, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the AXL transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:28, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_001265528.1 encoding the AXL polypeptide.

[0073] The term “AXL” also comprises nucleotide sequences showing a high degree of homology to AXL, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:27 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:28 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:28 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:27.

[0074] The term “NEXN” refers to the human nexilin F-actin binding protein gene (Ensembl: ENSG00000162614), for example, to the sequence as defined in NCBI Reference Sequence NM_144573.4, specifically, to the nucleotide sequence as set forth in SEQ ID NO:29, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the NEXN transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NQ:30, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_653174.3 encoding the NEXN polypeptide.

[0075] The term “NEXN” also comprises nucleotide sequences showing a high degree of homology to NEXN, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:29 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:30 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:30 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:29.

[0076] The term “CCDC80” refers to the human Coiled-coil domain-containing protein 80 gene (Ensembl: ENSG00000091986), for example, to the sequence as defined in NCBI Reference Sequence NM_199512.3, specifically, to the nucleotide sequence as set forth in SEQ ID NO:31 , which corresponds to the sequence of the above indicated NCBI Reference Sequences of the CCDC80 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:32, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_955805.1 encoding the CCDC80 polypeptide.

[0077] The term “CCDC80” also comprises nucleotide sequences showing a high degree of homology to CCDC80, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:31 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:32 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:32 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:31.

[0078] The term “FJX1” refers to the human Four jointed box 1 gene (Ensembl: ENSG00000179431), for example, to the sequence as defined in NCBI Reference Sequence NM_014344.4, specifically, to the nucleotide sequence as set forth in SEQ ID NO:33, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the FJX1 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:34, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_055159.2 encoding the FJX1 polypeptide.

[0079] The term “FJX1” also comprises nucleotide sequences showing a high degree of homology to FJX1 , e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:33 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:34 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:34 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:33.

[0080] The term “WWC1” refers to the human WW domain-containing protein 1 gene (Ensembl: ENSG00000113645), for example, to the sequence as defined in NCBI Reference Sequence NM_001161661.2, specifically, to the nucleotide sequence as set forth in SEQ ID NO:35, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the WWC1 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:36, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_001155133.1 encoding the WWC1 polypeptide.

[0081] The term “WWC1” also comprises nucleotide sequences showing a high degree of homology to WWCI , e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:35 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:36 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:36 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:35. The term “RASSF6” refers to the human Ras association domain family member 6 gene (Ensembl: ENSG00000169435), for example, to the sequence as defined in NCBI Reference Sequence NM_001270391 .2, specifically, to the nucleotide sequence as set forth in SEQ ID NO:37, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the RASSF6transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:38, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_001257320.1 encoding the RASSF6 polypeptide.

[0082] The term “RASSF6” also comprises nucleotide sequences showing a high degree of homology to RASSF6, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:37 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:38 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:38 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:37.

[0083] The term “NT5E” refers to the human 5'-nucleotidase gene (Ensembl: ENSG00000135318), for example, to the sequence as defined in NCBI Reference Sequence NM_002526.4, specifically, to the nucleotide sequence as set forth in SEQ ID NO:39, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the NT5E transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NQ:40, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_002517.1 encoding the NT5E polypeptide.

[0084] The term “NT5E” also comprises nucleotide sequences showing a high degree of homology to NT5E, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:39 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NQ:40 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:40 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:39.

[0085] The term “TEAD4” refers to the human Transcriptional enhancer factor TEF-3 gene (Ensembl: ENSG00000197905), for example, to the sequence as defined in NCBI Reference Sequence NM_003213.4, specifically, to the nucleotide sequence as set forth in SEQ ID NO:41 , which corresponds to the sequence of the above indicated NCBI Reference Sequences of the TEAD4 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:42, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_003204.2 encoding the TEAD4 polypeptide.

[0086] The term “TEAD4” also comprises nucleotide sequences showing a high degree of homology to TEAD4, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:41 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:42 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:42 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:42.

[0087] The term “S1 PR1” refers to the human Sphingosine-1-phosphate receptor 1 gene (Ensembl: ENSG00000170989), for example, to the sequence as defined in NCBI Reference Sequence NM_001400.5, specifically, to the nucleotide sequence as set forth in SEQ ID NO:43, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the S1 PR1 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:44, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_001391.2 encoding the S1 PR1 polypeptide. The term “S1 PR1” also comprises nucleotide sequences showing a high degree of homology to S1 PR1, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:43 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:44 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:44 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:43.

[0088] The term “NLIAK2” refers to the human NLIAK family SNF1-like kinase 2 gene (Ensembl: ENSG00000163545), for example, to the sequence as defined in NCBI Reference Sequence NM_030952.3, specifically, to the nucleotide sequence as set forth in SEQ ID NO:45, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the NLIAK2 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:46, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_112214.3 encoding the NLIAK2 polypeptide.

[0089] The term “NLIAK2” also comprises nucleotide sequences showing a high degree of homology to NLIAK2, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:45 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:46 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:46 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:45.

[0090] The term “KRAS” refers to the human Kirsten rat sarcoma virus gene (Ensembl: ENSG00000133703), for example, to the sequence as defined in NCBI Reference Sequence NM_004985.5, specifically, to the nucleotide sequence as set forth in SEQ ID NO:47, which corresponds to the sequence of the above indicated NCBI Reference Sequences of the KRAS transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:48, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_004976.2 encoding the KRAS polypeptide.

[0091] The term “KRAS” also comprises nucleotide sequences showing a high degree of homology to KRAS, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:47 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:48 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:48 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:47.

[0092] Thus in the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two or all twenty three, Hippo target genes are selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2. In an embodiment in the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or all fourteen, Hippo target genes are selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , and AXL. In an embodiment in the methods, products and uses broadly described herein the three or more, e.g. three, preferably six or more, four, five, six, seven, eight, nine, or all ten, Hippo target genes are selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , and CCN1. In an embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, Hippo target genes comprise one or more, for example one, two, three, four, five, six, seven, eight, or all nine, Hippo target genes selected from NPPB, CCN2, KISS1 , NEXN, S1 PR1 , SNAPCIx, SKP2, CCND1 , AXL, DKK1 , and ITGB2, and comprise one or more, for example one, two, three, four, five, or all six, Hippo target genes selected from IGFBP3, F3, FJX1 , NLIAK2, NT5E, and CCDC80.

[0093] In an embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, Hippo target genes comprise one or more, for example one, two, three, four, five, six, seven, eight, or all nine, Hippo target genes selected from NPPB, CCN2, KISS1 , NEXN, S1 PR1 , SNAPCIx, SKP2, CCND1 , AXL, DKK1 , and ITGB2, and comprise one or more, for example one, or both, Hippo target genes selected from RASSF6, and TEAD4.

[0094] In an embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, Hippo target genes comprise one or more, for example one, two, three, four, five, or all six, Hippo target genes selected from IGFBP3, F3, FJX1 , NLIAK2, NT5E, and CCDC, and comprise one or more, for example one, or both, Hippo target genes selected from RASSF6, and TEAD4.

[0095] In an embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, Hippo target genes comprise one or more, for example one, two, three, four, five, six, seven, eight, or all nine, Hippo target genes selected from NPPB, CCN2, KISS1 , NEXN, S1 PR1 , SNAPCIx, SKP2, CCND1 , AXL, DKK1 , and ITGB2, and comprise one or more, for example one, two, three, four, five, or all six, Hippo target genes selected from IGFBP3, F3, FJX1 , NUAK2, NT5E, and CCDC80, and comprise one or more, for example one, or both, Hippo target genes selected from RASSF6, and TEAD4.

[0096] The inventors further describe that various subsets of Hippo target genes successfully show differences in Hippo pathway activity for various datasets in which difference in Hippo pathway activity is expected. In particular, in Example 4 smaller subsets of Hippo target genes are used to determine Hippo pathway activity. In addition, Example 5 shows several six gene models supporting that small subsets such as a selection of six Hippo target genes could be used to accurately infer Hippo cellular signalling pathway activity, and for the methods and uses described herein (e.g. diagnostic or prognostic methods, monitoring, or determining treatment resistance), as these uses are all based on correctly inferring the Hippo pathway activity.

[0097] Therefore, in a further preferred embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two or all twenty three, Hippo target genes comprise or consist of one or more, e.g. one, two, three, four, five, six, seven, eight, nine, or all ten, Hippo target genes selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1. In a further preferred embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, or all ten, Hippo target genes are selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1.

[0098] In an additional embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two or all twenty three, Hippo target genes comprise or consist of one or more, e.g. one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen sixteen, seventeen, or all eighteen, Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, DKK1 , CCDC80, FJX1 , AXL, NEXN, TEAD4, and WWC1. In a further preferred embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen sixteen, seventeen, or all eighteen, Hippo target genes are selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, DKK1 , CCDC80, FJX1 , AXL, NEXN, TEAD4, and WWC1.

[0099] In an additional embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two or all twenty three, Hippo target genes comprise or consist of one or more, e.g. one, two, three, four, five, six, seven, eight, nine, ten, eleven twelve, thirteen or all fourteen, Hippo target genes selected from ANKRD1 , CCN2, F3, RASSF6, CCND1 , CCN1 , AMOTL2, NT5E, CCDC80, FJX1 , AXL, TEAD4, WWC1 , and NLIAK2. In a further preferred embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, ten, eleven twelve, thirteen or all fourteen, Hippo target genes are selected from ANKRD1 , CCN2, F3, RASSF6, CCND1 , CCN1 , AMOTL2, NT5E, CCDC80, FJX1 , AXL, TEAD4, WWC1 , and NUAK2.

[0100] In an additional embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two or all twenty three, Hippo target genes comprise or consist of one or more, e.g. one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or all fifteen, Hippo target genes selected from ANKRD1 , IGFBP3, CCN2, F3, CCND1 , CCN1 , AMOTL2, NT5E, ITGB3, CCDC80, FJX1 , TEAD4, S1 PR1 , WWC1 , and NLIAK2. In a further preferred embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or all fifteen, Hippo target genes are selected from ANKRD1 , IGFBP3, CCN2, F3, CCND1 , CCN1 , AMOTL2, NT5E, ITGB3, CCDC80, FJX1 , TEAD4, S1 PR1 , WWC1 , and NUAK2.

[0101] In a particularly preferred embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two or all twenty three, Hippo target genes comprise or consist of one or more, e.g. one, two, three, four, five, six, seven, eight, nine, or all ten, Hippo target genes selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1. In a further preferred embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, or all ten, Hippo target genes are selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1.

[0102] In a further preferred embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two or all twenty three, Hippo target genes comprise or consist of one or more, e.g. one, two, three, four, five, or all six, Hippo target genes selected from CCN2, F3, CCN1 , AMOTL2, TEAD4, and WWC1. In a further preferred embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, or all six, Hippo target genes are selected from CCN2, F3, CCN1 , AMOTL2, TEAD4, and WWC1.

[0103] In a further preferred embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two or all twenty three, Hippo target genes comprise or consist of one or more, e.g. one, two, three, four, five, or all six, Hippo target genes selected from ANKRD1 , CCND1 , AMOTL2, CCDC80, TEAD4, and WWC1. In a further preferred embodiment the methods, products and uses broadly described herein the three or more, preferably six or more, e.g. three, four, five, or all six, Hippo target genes are selected from ANKRD1 , CCND1 , AMOTL2, CCDC80, TEAD4, and WWCl .ln a more specific model, the Hippo cellular signaling pathway may be determined by (i) determining an activity level of a Hippo TF element in the sample, wherein the determining is based on evaluating a calibrated mathematical model relating the gene expression levels of three or more, preferably six or more, target genes of the Hippo cellular signaling pathway, the transcription of which is controlled by the Hippo TF element, to the activity level of the Hippo TF element, and by (ii) inferring the activity of the Hippo cellular signaling pathway in the sample based on the determined activity level of the Hippo TF element in the sample.

[0104] An “activity of a signaling pathway" may refer to the activity of a signaling pathway associated transcription factor (TF) element in the sample, the TF element controlling transcription of target genes, in driving the target genes to expression, i.e. , the speed by which the target genes are transcribed, e.g. in terms of high activity (i.e. high speed) or low activity (i.e. low speed), or other dimensions, such as levels, values or the like related to such activity (e.g. speed). Accordingly, for the purposes of the present invention, the term "activity", as used herein, is also meant to refer to an activity level that may be obtained as an intermediate result during “pathway analysis” as described herein. The term “transcription factor element” (TF element), as used herein, preferably refers to an intermediate or precursor protein or protein complex of the active transcription factor, or an active transcription factor protein or protein complex which controls the specified target gene expression. For example, the protein complex may contain at least the intracellular domain of one of the respective signaling pathway proteins, with one or more co-factors, thereby controlling transcription of target genes. Preferably, the term refers to either a protein or protein complex transcriptional factor triggered by the cleavage of one of the respective signaling pathway proteins resulting in a intracellular domain.

[0105] Herein, the term “Hippo transcription factor element” or “Hippo TF element” or “TF element” is defined to be a protein complex containing at least one of the proteins YAP or TAZ (also known as WWTR1), optionally with co-factors. Preferably, the term refers to either a protein or protein complex transcriptional factor, activated by binding a specific co-factor such as TEAD1 , TEAD2, TEAD3 or TEAD4. The Hippo TF element therefore preferably refers to a protein or protein complex comprising either one of YAP or TAZ wherein YAP or TAZ is not phosphorylated, and optionally one or more co-factors such as TEAD1 , TEAD2, TEAD3 or TEAD4 or a combination thereof. Optionally the Hippo TF element is further bound to an additional co-factor which may act as a transcription factor.

[0106] Therefore in an embodiment the inferring of the Hippo cellular signaling pathway activity is based on evaluating a calibrated mathematical pathway model, preferably the calibrated mathematical pathway model is a probabilistic model, more preferably a Bayesian network model, or wherein the mathematical pathway model is based on one or more (pseudo-)linear combination(s) of the gene expression levels of the three or more, preferably six or more, Hippo target genes.

[0107] In an embodiment the methods as broadly described herein further comprises a step of determining an activity level of a Hippo transcription factor (TF) element in the sample, the Hippo TF element controlling transcription of the three or more, preferably six or more, Hippo target genes, the determining being based on evaluating a calibrated mathematical pathway model relating the gene expression levels of the three or more, preferably six or more, Hippo target genes to the activity level of the Hippo TF element, and inferring the activity of the Hippo cellular signaling pathway in the sample based on the determined activity level of the Hippo TF element in the sample.

[0108] The calibrated mathematical pathway model may be a probabilistic model, preferably a Bayesian network model, based on conditional probabilities relating the activity level of the Hippo TF element and the gene expression levels of the three or more, preferably six or more, Hippo target genes, or the calibrated mathematical pathway model may be based on one or more linear combination(s) of the gene expression levels of the three or more, preferably six or more, Hippo target genes. In particular, the inferring of the activity of the Hippo cellular signaling pathway may be performed as disclosed in the published international patent application WO 2013 / 011479 A2 (“Assessment of cellular signaling pathway activity using probabilistic modeling of target gene expression”) or as described in the published international patent application WO 2014 / 102668 A2 (“Assessment of cellular signaling pathway activity using linear combination(s) of target gene expressions”), the contents of which are herewith incorporated in their entirety. Further details regarding the inferring of cellular signaling pathway activity using mathematical modeling of target gene expression can be found in Verhaegh W. et al., “Selection of personalized patient therapy through the use of knowledge-based computational models that identify tumor-driving signal transduction pathways”, Cancer Research, Vol. 74, No. 11 , 2014, pages 2936-2945.

[0109] The term “sample”, as used herein, refers to a specimen containing cells. The sample may be obtained from a tissue, a cell culture or a subject. The sample may be treated such as fixation by formalin. The term sample may also refer to a lysate of cells or its purified components, such as isolated RNA. When obtained from a subject the sample may for example be a biopsy or a blood sample.

[0110] The term “subject”, as used herein, refers to any living being. In some embodiments, the subject is an animal, preferably a mammal. In certain embodiments, the subject is a human being, preferably a medical subject.

[0111] The term “target gene” as used herein, means a gene whose transcription is directly or indirectly controlled by a Hippo transcription factor element. The “target gene” may be a “direct target gene” and / or an “indirect target gene” (as described herein). Preferably the target gene is a direct target gene. Particularly suitable Hippo target genes are described herein. In an embodiment the sample is obtained from a subject. In an embodiment the method for inferring activity of a Hippo cellular signaling pathway in a sample further comprises the step of obtaining the sample from the subject.

[0112] In an embodiment the methods as broadly described herein further comprise determining whether the Hippo cellular signaling pathway is operating at an undesired activity level in the sample based on the inferred activity of the Hippo cellular signaling pathway in the sample. The phrase “the cellular signaling pathway is operating abnormally” refers to the case where the “activity” of the pathway is not as expected, wherein the term “activity” may refer to the activity of the transcription factor complex in driving the target genes to expression, i.e. , the speed by which the target genes are transcribed. “Normal” may be when it is inactive in tissue where it is expected to be inactive or active where it is expected to be active. Consequently, “Abnormal” may be when it is inactive in tissue where it is expected to be active or active where it is expected to be inactive. Furthermore, there may be a certain level of activity that is considered “normal”, and anything higher or lower maybe considered “abnormal”. Thus abnormal activity may refer to activity which is lower than expected or refer to activity that is higher than expected.

[0113] By using a calibrated mathematical model to relate the gene expression levels to a cellular signaling pathway activity, a numerical value can be assigned to the pathway activity. This value can for example be normalized to result in a value from 0 to 100, where 0 is no pathway activity and 100 is the theoretical maximum pathway activity. Alternatively the value may be normalized such that the average value is 0 and thus decreased pathway activity is represented by a negative value and increased pathway activity is represented by a positive value. It is understood that the values obtained using such model are dependent on the model used, and do not represent absolute values. Therefore, the same model should be used for calibrating, determining reference values and when used in the method of the invention, so that it allows comparison of the obtained numerical values for pathway activities.

[0114] Therefore, the numerical value obtained for a pathway activity in a sample may be compared to the numerical value obtained for that pathway activity in a reference sample. By performing such comparison a statement can be made about the pathway activity in the sample (e.g. a tumor cell sample) relative to the pathway activity determined in the reference sample (e.g. a tumor cell sample with known resistance status, e.g. resistance to KRAS inhibitors). Based on the numerical value a statement can be made about the pathway activity in the sample with respect to the pathway activity in the reference sample, e.g. whether the pathway activity in the sample is higher or lower or similar, in correspondence with the numerical value obtained for the reference pathway activity. E.g. the Hippo cellular signaling pathway activity can be determined in a reference tumor cell sample, e.g. a reference tumor cell sample obtained from a patient being or to be treated with KRAS inhibitors. Since it is determined by the inventors that the Hippo cellular signaling pathway activity in a tumor cell sensitive to KRAS inhibitors generally is high, this can be set as a baseline numerical value representing an active pathway, i.e. a first reference value. In addition it is determined by the inventors that the Hippo cellular signaling pathway activity in a tumor cell resistant to KRAS inhibitors generally is low, this can be set as a baseline numerical value representing an inactive pathway, i.e. a second reference value. By determining the numerical value for the Hippo cellular signaling pathway activity in the sample to be analyzed, the numerical value representing that pathway activity can be compared with the reference value(s), and it can be determined whether the pathway activity is higher or equal (or lower) compared to the reference based on the numerical values. The comparison may be made with multiple reference samples to allow for more accurate results and statistics to be performed. Alternatively a reference sample can be used to set a baseline pathway activity, allowing further comparison of the pathway activity. For example, the average or mean and standard deviation can be calculated which can be used to calculate values for e.g. active and inactive Hippo pathway activities, and define a thresholds for abnormal pathway activity.

[0115] Therefore, in a preferred embodiment the pathway activity is determined to be higher or lower when the obtained numerical value for the pathway activity differs with at least one standard deviation from the numerical value obtained for the pathway activity in the reference sample. When the value is within the range of one standard it is said to be equal or comparable to the pathway activity in the reference sample, and consequently when it is one or more standard deviation higher the activity said to be “high” or “higher”. It is understood that the threshold may also be set higher, for example 2 times the standard deviation or even 3 times the standard deviation. It is further understood that the threshold may be determined using alternative ways, e.g. statistical methods, to determine a significant deviation from the reference value. Alternatively a preset value can be used as a cutoff for the activity of a pathway to be considered abnormal (with reference to the reference sample pathway activity). Therefore when used herein the relative terms “high” or “higher” and “low” or “lower” and “abnormal” in the context of cellular signaling pathway activity preferably refer to a predefined and validated pathway activity in a defined reference sample (or an average (or mean) of pathway activities determined in multiple defined reference samples).

[0116] It is understood that the reference value for a certain pathway activity in a sample in principle only needs to be determined once. Therefore, the step of determining a reference pathway activity is not necessarily an active part of the methods of the invention, as a predetermined reference pathway activity can be used.

[0117] Using a calibrated mathematical method to determine the cellular signaling pathway activity (e.g. based on the gene expression levels of target genes) thus allows the comparison between different samples. The mathematical model provides a numerical value for the determined pathway activity, which can be compared with the values obtained in one or more other samples. Thereby rendering it possible to use the method for different purposes, such as comparing the cellular signaling pathway activity with desired values (e.g. obtained in a validated reference sample or samples). The method can also be used to determine KRAS inhibitor sensitivity in cancer cells by comparing the Hippo pathway activity with Hippo pathway activity determined in reference tumor cells which or are not resistant to KRAS inhibitors.

[0118] By analyzing the determined pathway activity it can be determined whether the activity matches the expected pathway activity for the type of cell(s) being analyzed. This allows determining if a pathway activity is aberrant (e.g. too low or too high) by comparison to previously determined pathway activity in suitable reference samples. For example, to determine

[0119] The present invention also relates to a method (as described herein), wherein the normal or abnormal operation of the Hippo cellular signaling pathway is an operation in which the Hippo cellular signaling pathway operates as a tumor suppressor, an immune suppressor, or a driver of a disease or a disorder. In this respect it is recognized that normal activity of the Hippo cellular signaling pathway impedes cell proliferation and thus acts as a tumor suppressor. In this respect, in tumor cells abnormal activity of the Hippo pathway is observed in which low activity of the Hippo pathway allows cells to proliferate. Increasing Hippo pathway activity (restoring activity to normal levels) may contribute to reducing or inhibiting tumor growth.

[0120] The sample(s) to be used in accordance with the present invention may refer to any sample comprising one or more cells. Thus a sample may refer to a cell culture, 3D cell culture, engineered tissue, tissue culture, or organoid. A sample can also be an extracted sample, that is, a sample that has been extracted from a subject. Examples of the sample include, but are not limited to, a biopsy, a tissue, cells, blood and / or a body fluid of a subject. If the subject is a medical subject that has or may have cancer, it can be, e.g., a sample obtained from a cancer lesion, or from a lesion suspected for cancer, or from a metastatic tumor, or from a body cavity in which fluid is present which is contaminated with cancer cells (e.g., pleural or abdominal cavity or bladder cavity), or from other body fluids containing cancer cells, and so forth, preferably via a biopsy procedure or other sample extraction procedure. The cells of which a sample is extracted may also be tumorous cells from hematologic malignancies (such as leukemia or lymphoma). In some cases, the cell sample may also be circulating tumor cells, that is, tumor cells that have entered the bloodstream and may be extracted using suitable isolation techniques, e.g., apheresis or conventional venous blood withdrawal. If the subject is a medical subject that has or may have a different disease (meaning not cancer) the sample may be a sample obtained from a tissue relevant for the disease or representative for the disease. Aside from blood, a body fluid of which a sample is extracted may be urine, gastrointestinal contents, or an extravasate. The term “sample”, as used herein, also encompasses the case where e.g. a tissue and / or cells and / or a body fluid of the subject have been taken from the subject and, e.g., have been put on a microscope slide, and where for performing the claimed method a portion of this sample is extracted, e.g., by means of Laser Capture Microdissection (LCM), or by scraping off the cells of interest from the slide, or by fluorescence- activated cell sorting techniques. In addition, the term “sample”, as used herein, also encompasses the case where e.g. a tissue and / or cells and / or a body fluid of the subject have been taken from the subject and have been put on a microscope slide, and the claimed method is performed on the slide. In addition, the term “sample”, as used herein, also encompasses the case where e.g. a cell line and / or cell culture has been generated based on the cells / tissue / body fluid that have been taken from the subject. In an embodiment the methods broadly described herein further comprise recommending prescribing a drug for a subject that corrects for undesired activity level operation of the Hippo cellular signaling pathway, wherein the recommending is performed if the Hippo cellular signaling pathway is determined to be operating at an undesired activity level in the subject based on the inferred activity of the Hippo cellular signaling pathway. In an embodiment the undesired activity level is a too low activity level or a too high activity level. In an embodiment the undesired activity of the Hippo cellular signaling pathway is a too low activity and an activator of the Hippo pathway recommended. In an embodiment the undesired activity of the Hippo cellular signaling pathway is a too high activity and an inhibitor of the Hippo pathway recommended. In an embodiment the undesired activity level of the Hippo cellular signaling pathway is an activity level in which the Hippo cellular signaling pathway operates as a promoter of, or fails to inhibit, or is an indication of one or more of the following disorders: cancer, an eye disease, cardiac disease, pulmonary disease, renal disease, a central nervous system disorder, a hepatic disease, or an immune dysfunction.

[0121] In an embodiment the cancer is selected from bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, ependymoma, NF2-related Schwannomas, mesothelioma, and uveal melanoma. Preferably the cancer is selected from breast cancer, lung cancer, liver cancer, colorectal cancer, ependymoma, NF2-related Schwannomas, mesothelioma, and uveal melanoma. In an embodiment the eye disease is a retinal-related disease, such as retinal detachment or retinal detachment induced photoreceptor death. In an embodiment the eye disease is cataract. In an embodiment the cardiac disease is selected from cardiomyopathy, heart failure, coronary heart disease and myocardial infarction. In an embodiment the pulmonary disease is selected from COVID-19, pulmonary arterial hypertension, inflammatory pulmonary diseases, and pulmonary fibrosis. In an embodiment the central nervous system disorder is selected from a neurodegenerative diseases such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). In an embodiment the hepatic disease is selected from hepatitis and liver fibrosis. In an embodiment the immune dysfunction is selected from an impaired the innate immune response, an impaired adaptive immunity, or an autoimmune disease. Thus in an embodiment the methods and uses as broadly described herein further comprise a step of providing a treatment recommendation.

[0122] In a further embodiment the inferred Hippo cellular signaling pathway activity is used to predict an outcome for a subject. The outcome may be for a subject suffering from cancer, an eye disease, cardiac disease, pulmonary disease, renal disease, a central nervous system disorder, a hepatic disease, or an immune dysfunction as defined herein above. In an embodiment the inferred Hippo cellular signaling pathway activity is used to predict a treatment response for a subject.

[0123] In an embodiment the method describes determining the Hippo pathway activity based on previously determined (provided) target gene expression levels in a sample. Accordingly, in an embodiment the method is a computer implanted method. In an embodiment the method involves a step of determining the expression levels of three or more target genes in the sample. The "expression level of a target gene" denotes a value that is representative of the amount, e.g. a concentration, of a target gene present in a sample. This value may be based on the amount of a transcription product of the target gene (e.g. mRNA) or a translation product thereof (e.g. protein). Preferably, the expression level is based on the amount of mRNA formed from the target gene. In order to determine the expression level, techniques such as qPCR, multiple qPCR, multiplexed qPCR, ddPCR, RNAseq, RNA expression array or mass spectrometry may be used. For example, a gene expression microarray, e.g. Affymetrix microarray, or RNA sequencing methods, like an Illumina sequencer, can be used. Thus the provided or determine expression level may be based on qPCR, multiple qPCR, multiplexed qPCR, ddPCR, RNAseq, RNA expression array or mass spectrometry, among others.

[0124] In an embodiment the methods as broadly described herein for determining Hippo cellular signaling pathway activity are used to monitor a patient. For example by evaluating patients samples over time and analyzing those samples for Hippo cellular signaling pathway activity a patient may be monitored for treatment effect (e.g. effectiveness of a cancer therapy in a cancer patient), changes in disease (e.g. cancer becoming more aggressive or resistant to treatment, particularly GTPase and kinase inhibitor resistance), disease (e.g. cancer) progression or recurrence. In accordance with another disclosed aspect, an apparatus for inferring activity of the Hippo cellular signaling pathway in a sample comprises a digital processor configured to perform the method of the present invention as described herein.

[0125] In accordance with another disclosed aspect, a non-transitory storage medium for inferring activity of the Hippo cellular signaling pathway in a sample, stores instructions that are executable by a digital processing device to perform the method of the present invention as described herein. The non-transitory storage medium may be a computer- readable storage medium, such as a hard drive or other magnetic storage medium, an optical disk or other optical storage medium, a random access memory (RAM), read only memory (ROM), flash memory, or other electronic storage medium, a network server, or so forth. The digital processing device may be a handheld device (e.g., a personal data assistant or smartphone), a notebook computer, a desktop computer, a tablet computer or device, a remote network server, or so forth.

[0126] In accordance with another disclosed aspect, a computer program for inferring activity of the Hippo cellular signaling pathway in a sample comprises program code means for causing a digital processing device to perform the method of the present invention as described herein, when the computer program is run on the digital processing device. The digital processing device may be a handheld device (e.g., a personal data assistant or smartphone), a notebook computer, a desktop computer, a tablet computer or device, a remote network server, or so forth.

[0127] As described above the Hippo pathway plays a role in sensitivity and resistance to drugs, particularly GTPase and kinase inhibitors. As also shown in the experimental examples below, an active Hippo pathway is associated with cells being sensitive to inhibitors and an inactive (reduced active, lower active) Hippo pathway is associated with resistance to inhibitors. Thus the Hippo pathway activity can be used to determine, predict or monitor resistance to an inhibitor drug. Non limiting examples of inhibitors drugs are GTPase inhibitors such as but not limited to KRAS inhibitors, and kinase inhibitors such as but not limited to B-Raf inhibitors and MEK inhibitors.

[0128] Thus in a second aspect the invention relates to a method for determining or predicting resistance of a tumor to an inhibitor, the method comprising receiving the gene expression levels of three or more, preferably six or more, Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1, WWC1 , and NLIAK2, the gene expression levels of three or more, preferably six or more, Hippo target genes being determined in a sample obtained from the tumor; inferring the Hippo cellular signaling pathway activity based on the three or more, preferably six or more, gene expression levels, and predicting or determining the tumor to be or become resistant to inhibitors if the inferred Hippo cellular signaling pathway activity is low. In a preferred embodiment the three or more, preferably six or more, Hippo target genes comprise or consist of three or more (preferably six or more) Hippo target genes selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1.

[0129] When used herein the term inhibitor may refer to a GTPase inhibitor or a kinase inhibitor. The GTPase inhibitor may be a KRAS inhibitor. The KRAS inhibitor may be selected from Sotorasib (AMG 510), Adagrasib (MRTX849), GDC-6036, LY3499446, JNJ-74699157, D-1553, TNO155, MRTX1133, JDQ-443, RG6330, BPI-421286, GH35, BEBT-607 and JAB-21000. The kinase inhibitor may be a B-Raf inhibitor or a MEK inhibitor or a SHP2 inhibitor. The B-Raf inhibitor may be selected from Vemurafenib (RG7204 or PLX4032), GDC-0879, PLX-4720, Sorafenib, Dabrafenib and Encorafenib. The MEK inhibitor may be selected from Binimetinib (MEK162), Cobimetinib (XL518), Selumetinib, Trametinib (GSK1120212), PD-325901 , CI-1040, PD035901 and TAK-733. The SHP2 inhibitor may be selected from TNO155, JAB- 3068, RMC-4630, RLY-1971 and BBP-398. Additional information about the compounds including references can be found in Wu et al. ((2023) Small molecular inhibitors for KRAS-mutant cancers. Front. Immunol. 14:1223433) and Dunnett-Kane et al. (Mechanisms of Resistance to KRASG12C Inhibitors. Cancers 2021 , 13, 151). In a particularly preferred embodiment the invention describes a method for determining or predicting resistance of a tumor to a KRAS inhibitor, the method comprising receiving the gene expression levels of three or more, preferably six or more, Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2, the gene expression levels of three or more, preferably six or more, Hippo target genes being determined in a sample obtained from the tumor; inferring the Hippo cellular signaling pathway activity based on the three or more, preferably six or more, gene expression levels, and predicting or determining the tumor to be or become resistant to the KRAS inhibitor if the inferred Hippo cellular signaling pathway activity is low. In a preferred embodiment the three or more, preferably six or more, Hippo target genes comprise or consist of three or more (preferably six or more) Hippo target genes selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1. In an embodiment the KRAS inhibitor is selected from Sotorasib (AMG 510), Adagrasib (MRTX849), GDC-6036, LY3499446, JNJ-74699157, D-1553, TNO155, JAB-3068, RMC-4630, RLY-1971 , and BBP-398, preferably sotorasib.

[0130] The experimental results make plausible that Hippo cellular signaling pathway may be used to predict a tumor to become resistant to an inhibitor (e.g. a KRAS inhibitor), to determine if a tumor is resistant to an inhibitor, or to monitor a tumor for developing resistance to an inhibitor. Therefore, in an embodiment the predicting or determining is done prior to treatment onset or after onset of treatment with the inhibitor (e.g. a KRAS inhibitor). The method may further be used to recommend a treatment based on the observed Hippo pathway cellular signaling activity in the sample. For example, when Hippo pathway activity is high (the pathway is active) treatment with an inhibitor (e.g. a GTPase inhibitor such as a KRAS inhibitor or a kinase inhibitor) will likely be beneficial and thus treat (or continued treatment) with an inhibitor may be recommended, optionally supplemented with additional treatment options such as but not limited to a YAP inhibitor or Hippo activator to prevent the tumor from becoming resistant to the inhibitor. In a further example when Hippo pathway activity is low (the pathway is inactive) treatment with an inhibitor by itself (e.g. a GTPase inhibitor such as a KRAS inhibitor or a kinase inhibitor) will likely not be successful as the tumor is likely resistant to the inhibitor. In such case an alternative treatment may be recommended or a combination treatment of the inhibitor combined with a Hippo activator or YAP inhibitor to re-sensitize the tumor to the inhibitor. Thus in an embodiment the method comprises recommending a treatment. In an embodiment the recommended treatment is an inhibitor, such as a GTPase inhibitor or a kinase inhibitor if the inferred Hippo cellular signaling pathway activity is high. In an embodiment the recommended treatment is an alternative treatment if the inferred Hippo cellular signaling pathway activity is low. In an embodiment the alternative treatment is selected from chemotherapy, surgery, immunotherapy, radiation therapy, or combination therapy of the inhibitor (a GTPase inhibitor such as a KRAS inhibitor or a kinase inhibitor such as a MEK or a BRAF inhibitor) and an activator of the Hippo pathway or an inhibitor of an inhibitor of YAP and / or TAZ.

[0131] When used herein the terms activator of the Hippo pathway and inhibitor of YAP (or TAZ) are used interchangeably and refer to compounds or biologicals that increase the Hippo pathway activity resulting in the phosphorylation of YAP (and / or TAZ) and resulting decrease of YAP (TAZ) mediated transcriptional activity. Thus when referred herein a Hippo activator (or YAP / TAZ inhibitor) can be a compound that results in reduced transcription by the YAP / TAZ transcription complex. Thus such compound may act on a protein involved in the Hippo signaling cascade, such as but not limited to MST1 , MST2, SAV1 , LATS1 , LATS2, MOB1A, MOB1 B, TEAD1 , TEAD2, TEAD3, and TEAD4. Non limiting examples of Hippo activators or YAP (and / or TAZ) inhibitors as used herein are GNE-7883, (R)-1-(5-(4-Cyclohexylphenyl)-7-oxo-4,7- dihydropyrazolo[1 ,5-a]pyrimidine-3- carbonyl)pyrrolidine-3-carbonitrile (compound 2),

[0132] 5-(4-Cyclohexylphenyl)-3-(3-(fluoromethyl)azetidine-1-carbonyl)-2-(pyrazin-2- yl)pyrazolo[1 ,5-a]pyrimidin-7(4 / 7)-one (compound 3), Verteporfin, 9E1 , LPA, S1 P, thrombin, Epinephrine, glucagon, dihydrexidine, Dobutamine, Dasatinib, Latrunculin A, latrunculin B, cytochalasin D, Blebbistatin, ML7, Y27632, IAG933 (4-[(2S)-5-chloro-

[0133] 6-fluoro-2-phenyl-2-[(2S)-pyrrolidin-2-yl]-3H-1-benzofuran-4-yl]-5-fluoro-6-(2- hydroxyethoxy)-N-methylpyridine-3-carboxamide), K-975 (CAS 2563855-03-6), TM2 (CAS 1008768-41-9), VT-103 (CAS 2290608-13-6) and XMU-MMP-1 (CAS 2061980- 01-4). The term YAP / TAZ inhibitor may thus also refer to an inhibitor of TEAD1 , TEAD2, TEAD3, and / or TEAD4.

[0134] In an embodiment of the methods of the invention the method further comprises recommending the treatment using a combination of a inhibitor such as a KRAS inhibitor and a YAP / TAZ inhibitor if resistance to the inhibitor (e.g. a KRAS inhibitor) is predicted or determined in the tumor. In an embodiment the KRAS inhibitor is selected from Sotorasib (AMG 510), Adagrasib (MRTX849), GDC-6036, LY3499446, JNJ-74699157, D-1553, TNO155, JAB-3068, RMC-4630, RLY-1971 , and BBP-398, preferably sotorasib.

[0135] In an embodiment the tumor is bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, ependymoma, NF2-related Schwannomas, mesothelioma, and uveal melanoma. Preferably the tumor is selected from breast cancer, lung cancer, liver cancer, colorectal cancer, ependymoma, NF2-related Schwannomas, mesothelioma, and uveal melanoma. In an embodiment the tumor has a KRAS mutation. In an embodiment the KRAS mutation is a mutation of the Glycine at position 12 of human KRAS (position 12 of SEQ ID NO: 48), preferably wherein the mutation is a G12C, G12D or G12V mutation.

[0136] In an embodiment the Hippo cellular signaling pathway activity is determined according to the methods as broadly described herein.

[0137] In an embodiment the method provides the gene expression levels of the three or more, preferably six or more, Hippo target genes as determined in a sample, wherein the sample comprises a tumor cell obtained from the patient. Thus accordingly the method may be a computer implemented invention. Alternatively the method may comprise a step of providing a sample comprising at least one tumor cell from the subject and determining the expression levels of the three or more, preferably six or more, Hippo target genes in the sample. The sample may thus be a biopsy including liquid biopsies, a tissue sample, a stool sample,

[0138] Thus the method as broadly described herein can be used to determine existing resistance to inhibitors (such as KRAS inhibitors) in a tumor of a subject, or it can be used to predict if a tumor in a subject will develop resistance to inhibitors (such as KRAS inhibitors) prior to or during treatment with such inhibitors, or it can be used to monitor a subject with a tumor during treatment with inhibitors if the tumor of the subject becomes resistant to treatment with inhibitors. Accordingly, in an embodiment the method is used to predict if the tumor of a subject will become resistant to an inhibitor prior or during treatment with an inhibitor, is used to determine if the tumor of a subject is resistant to an inhibitor during treatment with an inhibitor, or to monitor if the tumor of a subject is or may become resistant to an inhibitor during treatment with an inhibitor. In an embodiment the inhibitor is a GTPase or kinase inhibitor as described herein, such as but not limited to a KRAS inhibitor.

[0139] The experimental data provided below (Example 2) further provides evidence that other signaling pathways are impacted in inhibitor resistance. In the data also presented in Figure 1 , it is shown that mock treated cells have higher MAPK, PI3K and NFkB cellular signaling pathway activities. When treated with an inhibitor such as a KRAS inhibitor MAPK, PI3K, and NFkB pathway activities are reduced. This makes sense as MAPK and PI3K are associated with proliferation and NFkB with cell survival. Upon successful treatment with an inhibitor such as a KRAS inhibitor these pathways are expected to be inhibited. It is observed that treating inhibitors resistant cells does not reduce the activities of these pathways, indicating that the treatment does not work and proliferation and cell survival pathways are not reduced upon inhibitor treatment. Therefore assessing these pathways in addition to the Hippo pathway may add useful information, as the MAPK and PI3K pathway activity will indicate if proliferation of the tumor is reduced or not and the NFkB pathway activity will indicate if cell survival pathways are reduced or not in the tumor. Accordingly in an embodiment the prediction is further based on determining one or more of the MAPK, PI3K and NFkB cellular signaling pathway activities. In an embodiment a high MAPK, PI3K and / or NFkB cellular signaling pathway activity corresponds to a tumor being resistant to an inhibitor and wherein a low MAPK, PI3K and / or NFkB cellular signaling pathway activity corresponds to a tumor being sensitive to an inhibitor. In an embodiment the the inhibitor is a GTPase or kinase inhibitor as described herein, such as but not limited to a KRAS inhibitor.

[0140] The MAPK, PI3K, WNT and NFkB pathway activities may be determined using methods previously described in WO 2019 / 120658 (MAPK), WO 2015 / 101635 (PI3K), WO 2013 / 011479 (WNT) and WO 2017 / 029215 (NFkB).

[0141] For example, the MAPK cellular signaling pathway may be determined by a computer- implemented method for inferring activity of a MAPK-AP-1 cellular signaling pathway in a sample performed by a digital processing device, wherein the inferring comprises: receiving gene expression levels of three or more target genes of the MAPK-AP-1 cellular signaling pathway measured in a sample, wherein the three or more AP-1 target genes are selected from the group consisting of: BCL2L11 , CCND1 , DDIT3, DNMT1 , EGFR, ENPP2, EZR, FASLG, FIGF, GLRX, IL2, IVL, LOR, MMP1 , MMP3, MMP9, SERPINE1 , PLAU, PLAUR, PTGS2, SNOG, TIMP1 , TP53, and VIM, preferably, from the group consisting of: CCND1 , EGFR, EZR, GLRX, MMP1 , MMP3, PLAU, PLAUR, SERPINE1 , SNOG, and TIMP1. In an embodiment determining an activity level of an AP-1 transcription factor (TF) element in the sample, the AP-1 TF element controlling transcription of the three or more AP-1 target genes, the determining being based on evaluating a calibrated mathematical pathway model relating the expression levels of the three or more AP-1 target genes to the activity level of the AP-1 TF element, and inferring the activity of the MAPK-AP-1 cellular signaling pathway in the sample based on the determined activity level of the AP-1 TF element in the sample. When used herein the terms “MAPK-AP-1” and “AP-1” are used interchangeable wherein the terms refer to the MAPK cellular signaling pathway controlled by the AP-1 transcription complex. AP-1 is a collective term for a group of dimeric transcription factors, primarily composed of proteins like c-Fos and c-Jun.

[0142] Herein, the term “AP-1 transcription factor element” or “AP-1 TF element” or “TF element” is defined to be a protein complex containing at least a member of the Jun (e.g. c-Jun, JunB and JunB) family and / or a member of the Fos (e.g. c-Fos, FosB, Fra- I and Fra-2) family and / or a member of the ATF family and / or a member of the JDP family, forming e.g. Jun-Jun or Jun~Fos dimers, capable of binding to specific DNA sequences, preferably the response elements 12-0-Tetradecanoylphorbol-13-acetate (TPA) response element (TRE) with binding motif 5’-TGA G / C TCA-3’ or cyclic AMP response element (CRE) with binding motif 5’-TGACGTCA-3’, thereby controlling transcription of target genes.

[0143] Preferably, the term refers to either a protein or protein complex transcriptional factor triggered by the binding of AP-1 inducing ligands, such as growth factors (e.g., EGF) and cytokines, to its receptor or an intermediate downstream signaling agent, or triggered by the presence of an AP1-activating mutation.

[0144] The calibrated mathematical pathway model may be a probabilistic model, preferably a Bayesian network model, based on conditional probabilities relating the activity level of the AP-1 TF element and the 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 combination(s) of the expression levels of the three or more AP-1 target genes. In particular, the inferring of the activity of the MAPK-AP-1 cellular signaling pathway may be performed as disclosed in the published international patent application WO 2013 / 011479 A2.

[0145] In a further example the PI3K cellular signaling pathway may be determined by a computer implemented method comprising inferring activity of a PI3K cellular signaling pathway in a sample subject based at least on receiving measured expression levels of three or more target genes of the PI3K cellular signaling pathway in the sample, wherein the three or more PI3K target genes are selected from the group consisting of: AGRP, BCL2L11 , BCL6, BNIP3, BTG1 , CAT, CAV1 , CCND1 , CCND2, CCNG2, CDKN1A, CDKN1 B, ESR1, FASLG, FBXO32, GADD45A, INSR, MXI1 , NOS3, PCK1 , POMC, PPARGC1A, PRDX3, RBL2, SOD2 and TNFSF10. In an embodiment the inferring comprises determining a level of a FOXO transcription factor (TF) element in the sample, the FOXO TF element controlling transcription of the three or more target genes of the PI3K cellular signaling pathway, the determining being based at least in part on evaluating a mathematical model relating expression levels of the three or more target genes of the PI3K cellular signaling pathway to the level of the FOXO TF element; inferring the activity of the PI3K cellular signaling pathway in the sample based on the determined level of the FOXO TF element in the sample. Herein, a FOXO transcription factor (TF) element is defined to be a protein complex containing at least one of the FOXO TF family members, i.e., FOXO1 , FOXO3A, FOXO4 and FOXO6, which is capable of binding to specific DNA sequences, thereby controlling transcription of target genes.

[0146] In a further example, the WNT cellular signaling pathway activity may be determined by a computer implemented method comprising inferring activity of an WNT cellular signaling pathway based at least on expression levels of three or more target genes of the WNT cellular signaling pathway measured in a sample, wherein the three or more target genes are selected from the group consisting of: KIAA1199, AXIN2, RNF43, TBX3, TDGF1 , SOX9, ASCL2, IL8, SP5, ZNRF3, KLF6, CCND1 , DEFA6 and FZD7, or three or more target genes selected from the group comprising or consisting of: NKD1 , OAT, FAT1 , LEF1 , GLUL, REG1 B, TCF7L2, COL18A1 , BMP7, SLC1A2, ADRA2C, PPARG, DK 1 , HNF1 A and LECT2. In an embodiment wherein the inferring comprises determining a level of an WNT transcription factor (TF) element in the sample, the WNT TF element controlling transcription of the three or more target genes of the WNT cellular signaling pathway, the determining being based at least in part on evaluating a mathematical model relating expression levels of the three or more target genes of the WNT cellular signaling pathway to the level of the WNT TF element, inferring the activity of the WNT cellular signaling pathway based on the determined level of the WNT TF element in the sample. Herein, a WNT transcription factor (TF) element is defined to be a protein complex containing at least beta-Catenin or preferably a complex comprising beta-Catenin and TCF and / or LEF, which is capable of binding to specific DNA sequences, thereby controlling transcription of target genes. In a further example, the NFkB cellular signaling pathway activity may be determined by a computer implemented method comprising inferring activity of an NFkB cellular signaling pathway based at least on expression levels of three or more target genes of the NFkB cellular signaling pathway measured in a sample, wherein the three or more target genes are selected from the group consisting of: BCL2L1 , BIRC3, CCL2, CCL3, CCL4, CCL5, CCL20, CCL22, CX3CL1 , CXCL1 , CXCL2, CXCL3, ICAM1 , IL1 B, IL6, IL8, IRF1 , MMP9, NFKB2, NFKB1A, NFKB1 E, PTGS2, SELE, STAT5A, TNF, TNFAIP2, TNIP1 , TRAF1 , and VCAM1. In an embodiment wherein the inferring comprises determining a level of an NFkB transcription factor (TF) element in the sample, the NFkB TF element controlling transcription of the three or more target genes of the NFkB cellular signaling pathway, the determining being based at least in part on evaluating a mathematical model relating expression levels of the three or more target genes of the NFkB cellular signaling pathway to the level of the NFkB TF element, inferring the activity of the NFkB cellular signaling pathway based on the determined level of the NFkB TF element in the sample. Herein, an NFkB transcription factor (TF) element is defined to be a protein complex containing at least one or, preferably, a dimer of the NFkB members (NFKB1 or p50 / p105, NFKB2 or p52 / p100, RELA or p65, REL, and RELB), which is capable of binding to specific DNA sequences, thereby controlling transcription of target genes.

[0147] Thus in an aspect the invention relates to a method of reducing resistance of a cell to an inhibitor by treating the cell with a YAP / TAZ inhibitor. The invention thus describes a YAP / TAZ inhibitor for use in the treatment, prevention or amelioration of a disease, the use comprising administering the YAP / TAZ inhibitor to a subject in need thereof thereby reducing resistance to the inhibitor. Alternatively the invention describes a method of treatment, prevention or amelioration of a disease by administering a YAP / TAZ inhibitor to a subject in need thereof, thereby reducing inhibitor resistance. In an embodiment the method or use involves inferring the Hippo cellular signaling pathway activity as broadly described herein. In an embodiment the disease is cancer, preferably bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, ependymoma, NF2- related Schwannomas, mesothelioma, or uveal melanoma, more preferably breast cancer, lung cancer, liver cancer, colorectal cancer, ependymoma, NF2-related Schwannomas, mesothelioma, or uveal melanoma. In an embodiment the inhibitor is a GTPase inhibitor, preferably a KRAS inhibitor, more preferably a KRAS inhibitor is selected from Sotorasib (AMG 510), Adagrasib (MRTX849), GDC-6036, LY3499446, JNJ-74699157, D-1553, TNO155, JAB-3068, RMC-4630, RLY-1971 , and BBP-398, preferably sotorasib. In an embodiment the inhibitor is a kinase inhibitor, preferably a B-Raf inhibitor or a MEK inhibitor or a SHP2 inhibitor. In an embodiment the B-Raf inhibitor is selected from Vemurafenib (RG7204 or PLX4032), GDC-0879, PLX-4720, Sorafenib, Dabrafenib and Encorafenib. In an embodiment the MEK inhibitor is selected from Binimetinib (MEK162), Cobimetinib (XL518), Selumetinib, Trametinib (GSK1120212), PD-325901 , CI-1040, PD035901 and TAK-733. In an embodiment the SHP2 inhibitor may be selected from TNO155, JAB-3068, RMC-4630, RLY-1971 and BBP-398. In an embodiment the YAP / TAZ inhibitor is selected from GNE-7883, (R)-1- (5-(4-Cyclohexylphenyl)-7-oxo-4,7-dihydropyrazolo[1 ,5-a]pyrimidine-3- carbonyl)pyrrolidine-3-carbonitrile (compound 2), 5-(4-Cyclohexylphenyl)-3-(3- (fluoromethyl)azetidine-1-carbonyl)-2-(pyrazin-2-yl)pyrazolo[1 , 5-a]pyrimidin-7(4 / 7)- one (compound 3), Verteporfin, 9E1 , LPA, S1 P, thrombin, Epinephrine, glucagon, dihydrexidine, Dobutamine, Dasatinib, Latrunculin A, latrunculin B, cytochalasin D, Blebbistatin, ML7, Y27632, IAG933 (4-[(2S)-5-chloro-6-fluoro-2-phenyl-2-[(2S)- pyrrolidin-2-yl]-3H-1-benzofuran-4-yl]-5-fluoro-6-(2-hydroxyethoxy)-N-methylpyridine- 3-carboxamide), K-975 (CAS 2563855-03-6), TM2 (CAS 1008768-41-9), VT-103 (CAS 2290608-13-6) and XMU-MMP-1 (CAS 2061980-01-4).

[0148] In a further aspect the invention relates to an inhibitor for use in the treatment, prevention or amelioration of cancer in a subject, the use comprising receiving the gene expression levels of three or more, preferably six or more, Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2, the gene expression levels of three or more, preferably six or more, Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the three or more, preferably six or more, gene expression levels, and administering the inhibitor when the Hippo pathway is active. In a preferred embodiment the three or more, preferably six or more, Hippo target genes comprise or consist of three or more (preferably six or more) Hippo target genes selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1. In an embodiment the inhibitor may be a GTPase inhibitor or a kinase inhibitor. The GTPase inhibitor may be a KRAS inhibitor. The KRAS inhibitor may be selected from Sotorasib (AMG 510), Adagrasib (MRTX849), GDC-6036, LY3499446, JNJ-74699157, D-1553, TNO155, MRTX1133, JDQ-443, RG6330, BPI-421286, GH35, BEBT-607 and JAB-21000. The kinase inhibitor may be a B-Raf inhibitor or a MEK inhibitor or a SHP2 inhibitor. The B-Raf inhibitor may be selected from Vemurafenib (RG7204 or PLX4032), GDC-0879, PLX-4720, Sorafenib, Dabrafenib and Encorafenib. The MEK inhibitor may be selected from Binimetinib (MEK162), Cobimetinib (XL518), Selumetinib, Trametinib (GSK1120212), PD-325901 , CI-1040, PD035901 and TAK-733. The SHP2 inhibitor may be selected from TNO155, JAB-3068, RMC-4630, RLY-1971 and BBP-398.

[0149] In an embodiment the cancer is selected from bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, ependymoma, NF2-related Schwannomas, mesothelioma, and uveal melanoma. Preferably the cancer is selected from breast cancer, lung cancer, liver cancer, colorectal cancer, ependymoma, NF2-related Schwannomas, mesothelioma, and uveal melanoma. In a further embodiment the inhibitor is combined with a YAP / TAZ inhibitor. Doing so may increase sensitivity and / or avoid resistance to the inhibitor.

[0150] In a further aspect the invention relates to a YAP / TAZ inhibitor for use in the treatment, prevention or amelioration of cancer in a subject, the use comprising receiving the gene expression levels of three or more, preferably six or more, Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2, the gene expression levels of three or more, preferably six or more, Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the three or more, preferably six or more, gene expression levels, and administering the YAP / TAZ inhibitor when the Hippo pathway is inactive. In a preferred embodiment the three or more, preferably six or more, Hippo target genes comprise or consist of three or more (preferably six or more) Hippo target genes selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1. In an embodiment the YAP / TAZ inhibitor is selected from GNE-7883, (R)-1-(5-(4- Cyclohexylphenyl)-7-oxo-4,7-dihydropyrazolo[1 ,5-a]pyrimidine-3- carbonyl)pyrrolidine-3-carbonitrile (compound 2),5-(4-Cyclohexylphenyl)-3-(3- (fluoromethyl)azetidine-1-carbonyl)-2-(pyrazin-2-yl)pyrazolo[1 , 5-a]pyrimidin-7(4 / 7)- one (compound 3), Verteporfin, 9E1 , LPA, S1 P, thrombin, Epinephrine, glucagon, dihydrexidine, Dobutamine, Dasatinib, Latrunculin A, latrunculin B, cytochalasin D, Blebbistatin, ML7, Y27632, IAG933 (4-[(2S)-5-chloro-6-fluoro-2-phenyl-2-[(2S)- pyrrolidin-2-yl]-3H-1-benzofuran-4-yl]-5-fluoro-6-(2-hydroxyethoxy)-N-methylpyridine- 3-carboxamide), K-975 (CAS 2563855-03-6), TM2 (CAS 1008768-41-9), VT-103 (CAS 2290608-13-6) and XMU-MMP-1 (CAS 2061980-01-4).

[0151] Thus in an embodiment the invention relates to a Hippo activator for use in the treatment, prevention or amelioration of cancer in a subject, the use comprising receiving the gene expression levels of three or more, preferably six or more, Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2, the expression levels of three or more, preferably six or more, Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the three or more, preferably six or more, gene expression levels, and administering the Hippo activator when the Hippo pathway is inactive. In a preferred embodiment the three or more, preferably six or more, Hippo target genes comprise or consist of three or more (preferably six or more) Hippo target genes selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1.

[0152] Accordingly in a further aspect the invention describes a method of treating, preventing or ameliorating cancer in a subject in need thereof, the method comprising: receiving the gene expression levels of three or more, preferably six or more, Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2, the expression levels of three or more, preferably six or more, Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the three or more, preferably six or more, gene expression levels, and if the Hippo pathway is active administering an inhibitor to the subject; or if the Hippo pathway is inactive administering a Hippo activator optionally combined with an inhibitor, a YAP / TAZ inhibitor optionally combined with an inhibitor or an alternative therapy selected from chemotherapy, surgery, immunotherapy, radiation therapy, a combination therapy of the inhibitor and a YAP / TAZ inhibitor, or a combination these therapies. In an embodiment the cancer is selected from bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, ependymoma, NF2-related Schwannomas, mesothelioma, and uveal melanoma. Preferably the cancer is selected from breast cancer, lung cancer, liver cancer, colorectal cancer, ependymoma, NF2-related Schwannomas, mesothelioma, and uveal melanoma. In an embodiment the inhibitor is a GTPase inhibitor or a kinase inhibitor. The GTPase inhibitor may be a KRAS inhibitor. The KRAS inhibitor may be selected from Sotorasib (AMG 510), Adagrasib (MRTX849), GDC-6036, LY3499446, JNJ-74699157, D-1553, TNO155, MRTX1133, JDQ-443, RG6330, BPI-421286, GH35, BEBT-607 and JAB-21000. The kinase inhibitor may be a B-Raf inhibitor or a MEK inhibitor or a SHP2 inhibitor. The B-Raf inhibitor may be selected from Vemurafenib (RG7204 or PLX4032), GDC-0879, PLX-4720, Sorafenib, Dabrafenib and Encorafenib. The MEK inhibitor may be selected from Binimetinib (MEK162), Cobimetinib (XL518), Selumetinib, Trametinib (GSK1120212), PD-325901 , CI-1040, PD035901 and TAK-733. The SHP2 inhibitor may be selected from TNO155, JAB-3068, RMC-4630, RLY-1971 and BBP-398. In an embodiment the YAP / TAZ inhibitor is selected from GNE-7883, (R)-1-(5-(4-Cyclohexylphenyl)-7-oxo-4,7- dihydropyrazolo[1 ,5-a]pyrimidine-3- carbonyl)pyrrolidine-3-carbonitrile (compound 2),

[0153] 5-(4-Cyclohexylphenyl)-3-(3-(fluoromethyl)azetidine-1-carbonyl)-2-(pyrazin-2- yl)pyrazolo[1 ,5-a]pyrimidin-7(4 / 7)-one (compound 3), Verteporfin, 9E1 , LPA, S1 P, thrombin, Epinephrine, glucagon, dihydrexidine, Dobutamine, Dasatinib, Latrunculin A, latrunculin B, cytochalasin D, Blebbistatin, ML7, Y27632, IAG933 (4-[(2S)-5-chloro-

[0154] 6-fluoro-2-phenyl-2-[(2S)-pyrrolidin-2-yl]-3H-1-benzofuran-4-yl]-5-fluoro-6-(2- hydroxyethoxy)-N-methylpyridine-3-carboxamide), K-975 (CAS 2563855-03-6), TM2 (CAS 1008768-41-9), VT-103 (CAS 2290608-13-6) and XMU-MMP-1 (CAS 2061980-

[0155] In a preferred embodiment the three or more, preferably six or more, Hippo target genes comprise or consist of three or more (preferably six or more) Hippo target genes selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1.

[0156] In a further aspect the invention relates to a kit of parts comprising polymerase chain reaction primers and optionally probes for specifically determining the gene expression levels of three or more, preferably six or more, Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2. In a preferred embodiment the three or more, preferably six or more, Hippo target genes comprise or consist of three or more (preferably six or more) Hippo target genes selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1. In an embodiment the kit further includes the apparatus or the non-transitory storage medium as broadly described herein.

[0157] The invention further relates to the use of the kit as broadly described herein in inferring the Hippo cellular signaling pathway activity, the use comprising determining the gene expression levels of three or more, preferably six or more, Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2 using the polymerase chain reaction primers and optionally probes provided in the kit and inferring the Hippo cellular signaling pathway based on the gene expression levels of the three or more, preferably Hippo target genes. In an embodiment the Hippo cellular signaling pathway is determined using the method as broadly described herein. In an embodiment the determining of the gene expression levels with the primers and probes involves the specific amplification and detection of the Hippo target genes. In a preferred embodiment the three or more, preferably six or more, Hippo target genes comprise or consist of three or more (preferably six or more) Hippo target genes selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1.

[0158] This application describes several preferred embodiments. Modifications and alterations may occur to others upon reading and understanding the preceding detailed description. It is intended that the application is construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof. Thus it shall be understood that the method as broadly described herein, the apparatus as broadly described herein, the non-transitory storage medium as broadly described herein, the computer program as broadly described herein, the compounds for use as broadly described herein, the kits as broadly described herein, and the use of the kits as broadly described herein have similar and / or identical preferred embodiments, in particular, as defined in the dependent claims.

[0159] It shall be understood that a preferred embodiment of the present invention can also be any combination of the dependent claims or above embodiments with the respective independent claim.

[0160] These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

[0161] Overview of mentioned genes and proteins not listed in the sequence listing: name reference

[0162] MST1 (STK4) ENSG00000173531

[0163] MST2 (STK3) ENSG00000104375

[0164] LATS1 ENSG00000131023

[0165] LATS2 ENSG00000150457

[0166] YAP (YAP1) ENSG00000137693

[0167] TAZ (WWTR1) ENSG00000018408

[0168] TEAD1 ENSG00000187079

[0169] TEAD2 ENSG00000074219

[0170] TEAD3 ENSG00000007866

[0171] TEAD4 ENSG00000197905

[0172] Examples

[0173] Example 1 - testing and validating Hippo pathway model.

[0174] A preliminary Hippo pathway model was constructed based on the putative Hippo target genes ANKRD1, CCN1 , NPPB, CCN2, KISS1 , NEXN, PAWR, S1 PR1 , SNAPC1 , SKP2, CCND1 , AXL, DKK1 , ITGB2, WWC1 , YAP, and TEAD1. The pathway model was tested and validated on the ground truth datasets GSE92335, and GSE10196. The data are shown below:

[0175] GSE92335 comprises transcription data from human colon cancer cell line transfected with control or YAP1 targeting siRNA. YAP1 activity is repressed by an active Hippo pathway, so its targeted degradation implies increased Hippo activity. The experiments where done in triplicate. The dataset is described in https: / / www.ncbi. nlm.nih.gov / geo / query / acc.cgi?acc=GSE92335 and Ou et al. MiR- 590-5p, a density-sensitive microRNA, inhibits tumorigenesis by targeting YAP1 in colorectal cancer. Cancer Lett. 2017 Jul 28;399:53-63. sample description Hippo activity

[0176] GSM2427772 HCT116 control siRNA 43

[0177] GSM2427773 HCT116 control siRNA 38

[0178] GSM2427774 HCT116 control siRNA 41

[0179] GSM2427775 HCT116 YAP siRNA 67

[0180] GSM2427776 HCT116 YAP siRNA 69

[0181] GSM2427777 HCT116 YAP siRNA 71

[0182] GSE10196 comprises transcription data from normal human mammary cell line MCF10A transfected with control or YAP over expressing cells. Over expression is assumed to reflect low Hippo pathway activity. The experiments where done in duplicate. The dataset is described in https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE10196 and Zhang et al. Negative regulation of YAP by LATS1 underscores evolutionary conservation of the Drosophila Hippo pathway. Cancer Res. 2008 Apr 15;68(8):2789-94. sample description Hippo activity

[0183] GSM257525 MCF10A control 36

[0184] GSM257527 MCF10A control 44

[0185] GSM257526 MCF10A YAP overexpression 33

[0186] GSM257528 MCF10A YAP overexpression 32 The results indicate that the pathway model is effective as it shows increased pathway activity upon YAP inhibition and slightly decreased pathway activity upon YAP overactivation. The dataset is described in

[0187] Example 2 - predicting KRAS inhibitor resistance

[0188] The preliminary Hippo pathway test was applied to public datasets where an KRAS inhibitor was applied to cells harboring a KRAS G12C mutation which are either sensitive or resistant to the KRAS inhibitor.

[0189] GSE229071 comprises transcription data from Non-Small Cell Lung Carcinoma cell lines with KRAS G12C mutation (NCI-H358 and NCI-H23) treated with the KRAS inhibitor AMG510 (Sotorasib) or control (DMSO). The dataset includes data from cell lines rendered resistant to the inhibitor by prolonged exposure to the inhibitor (indicated with “R” below). The dataset is described in https: / / www.ncbi. nlm.nih.gov / geo / query / acc.cgi?acc=GSE229071 and Hagenbeek et al. An allosteric pan-TEAD inhibitor blocks oncogenic YAP / TAZ signaling and overcomes KRAS G12C inhibitor resistance. Nat Cancer. 2023 Jun;4(6):812-828. doi: 10.1038 / S43018-023-00577-0. sample description Hippo activity

[0190] SRR24804864 NCI-H358 DMSO 51

[0191] SRR24804863 NCI-H358 DMSO 53

[0192] SRR24804862 NCI-H358 DMSO 54

[0193] SRR24804861 NCI-H358 AMG510 55

[0194] SRR24804860 NCI-H358 AMG510 53

[0195] SRR24804859 NCI-H358 AMG510 52

[0196] SRR24804858 NCI-H358-R AMG510 17

[0197] SRR24804857 NCI-H358-R AMG510 16

[0198] SRR24804856 NCI-H358-R AMG510 16 sample description Hippo activity

[0199] SRR24804855 NCI-H23 DMSO 60

[0200] SRR24804854 NCI-H23 DMSO 53 SRR24804853 NCI-H23 AMG510 64

[0201] SRR24804852 NCI-H23 AMG510 61

[0202] SRR24804851 NCI-H23-R AMG510 33

[0203] SRR24804850 NCI-H23-R AMG510 35

[0204] The results indicate that Hippo pathway activity is severely reduced in KRAS inhibitor resistant cells. The results are also depicted in Figure 1 , along with information on other pathways (MAPK, PI3K, NFkB, and WNT). As expected MAPK and PI3K pathway activities are high in untreated cells, indicating strong proliferation of the cells. MAPK and PI3K pathway activities are lowered upon treating the cells with a KRAS inhibitor, indicating reduced proliferation. Treating KRAS inhibitor resistant cells with a KRAS inhibitor does not reduce MAPK and PI3K pathway activities, indicating that proliferation remains unaffected (high) and further signifying that Hippo pathway activity is indicative of KRAS resistance.

[0205] Example 3 - predicting YAP / TAZ inhibitor treatment success

[0206] Next the Hippo pathway activity was assessed on a public dataset comprising samples describing untreated or treated samples with (putative) YAP (TEAD) inhibitors.

[0207] GSE229066 comprises transcription data from an ovarian cancer cell line (0VCAR8) and adrenal cortex carcinoma cell line (HCC1576) mock treated or treated with TEAD inhibitor GNE-7883 or putative TEAD inhibitors (compounds 2, 3, and 4). TEAD functions as a cofactor of YAP. The data are described in https: / / www.ncbi. nlm.nih.gov / geo / query / acc.cgi?acc=GSE229071 and Hagenbeek et al. An allosteric pan-TEAD inhibitor blocks oncogenic YAP / TAZ signaling and overcomes KRAS G12C inhibitor resistance. Nat Cancer. 2023 Jun;4(6):812-828. doi: 10.1038 / S43018-023-00577-0. sample

[0208] SRR24085127 HCC1576 DMSO

[0209] SRR24085128 HCC1576 DMSO 8

[0210] SRR24085129 HCC1576 DMSO 8

[0211] SRR24085130 HCC1576 GNE-7883 40 SRR24085131 HCC1576 GNE-7883 41

[0212] SRR24085132 HCC1576 GNE-7883 40

[0213] SRR24085133 HCC1576 Compound 3 38

[0214] SRR24085134 HCC1576 Compound 3 38

[0215] SRR24085135 HCC1576 Compound 3 37

[0216] SRR24085136 HCC1576 Compound 2 29

[0217] SRR24085137 HCC1576 Compound 2 30

[0218] SRR24085138 HCC1576 Compound 2 29

[0219] SRR24085139 HCC1576 Compound 4 8

[0220] SRR24085140 HCC1576 Compound 4 8

[0221] SRR24085141 HCC1576 Compound 4 9

[0222] The data show that inhibiting TEAD / YAP increases Hippo signaling pathway activity, and further shows that compounds 2 and 3 but not compound 4 have the same effect. The data makes plausible that YAP / TEAD inhibitors may be used to increase or restore Hippo activity, for example to reduce or revert sensitivity to inhibitors such as KRAS inhibitors. The data further shows that the Hippo pathway model allows for screening of compounds having an inhibitory effect on YAP and / or TEAD.

[0223] The results are also depicted in Figure 2, along with information on other pathways (MAPK, PI3K, NFkB, and WNT). As expected the PI3K pathway activities are high in untreated cells, indicating strong proliferation of the cells. PI3K pathway activities are lowered upon treating the cells with TEAD inhibitors, indicating reduced proliferation. Treating cells with a compound that does not increase Hippo activity (compound 4) does not result in reduced PI3K pathway activity, indicating that proliferation remains unaffected (high).

[0224] Further validation and fine tuning of the Hippo pathway activity model

[0225] Although reasonably good results were obtained the Hippo pathway target genes are all sourced from a single source (Stein et al. (2015) YAP1 Exerts Its Transcriptional Control via TEAD-Mediated Activation of Enhancers. PLoS Genet 11(8): e1005465.). This source specifically identifies target genes in cancer (glioblastoma, mesothelioma) which may introduce a selection bias and may miss other relevant target genes needed for a general pathway model (as in not specifically tailored for specific cancer types). Therefore other sources were reviewed (Wang Y, et al.; Cancer Genome Atlas Research Network; Camargo F, Liang H. Comprehensive Molecular Characterization of the Hippo Signaling Pathway in Cancer. Cell Rep. 2018 Oct 30;25(5):1304-1317.e5; and Wang C et al. RHPCG: a database of the Regulation of the Hippo Pathway in Cancer Genome, Database, Volume 2019, 2019, baz135) to source other potential target genes.

[0226] The change of expression levels of all target genes from the above three referenced sources were reviewed in the four above described experiments (12 datapoints each).

[0227] The changes of individual target gene expression levels over the 12 datapoints were combined in a “Proofpoint score” which ranks changes expression levels in the datapoints. The Proofpoint score is calculated be adding up the absolute values of the differences of expression levels (based on normalized expression levels) for each Proofpoint per target gene. The ranking is provided below:

[0228] Based on this ranking a Proofpoint score cutoff value of 8 was used as a final selection of target genes for the Hippo pathway model: NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AM0TL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1, WWC1 , and NUAK2.

[0229] The final model was tested and validated on the same datasets, and improved accuracy as predicted. In addition random selections of six genes were tested and demonstrate that a minimum of six target genes from the curated list suffice for a pathway model. This is not unexpected as the top scoring genes of the list are predictive of Hippo pathway activity in most datapoints already individually.

[0230] From this it is concluded that based on any selection of three target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2 the Hippo cellular signaling pathway activity can be accurately determined in different sample types. e 4 Reducing the number of genes in the pathway model

[0231] In order to reduce the number of genes in the pathway model different model with number of target genes were calibrated on distinct calibration sets as follows:

[0232] Models 1A (18 genes), 1 B (18 genes), 2A (14 genes), and 2B (15 genes) were calibrated on different cancer lines with or without two distinct inhibitors. Models 1A and 1 B are based on the target genes NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, DKK1 , CCDC80, FJX1 , AXL, NEXN, TEAD4, WWC1 (but with different weights).

[0233] Model 2A is based on the target genes ANKRD1 , CCN2, F3, RASSF6, CCND1 , CCN1 , AMOTL2, NT5E, CCDC80, FJX1 , AXL, TEAD4, WWC1 , NUAK2.

[0234] Model 2B is based on the target genes ANKRD1 , IGFBP3, CCN2, F3, CCND1 , CCN1 , AMOTL2, NT5E, ITGB3, CCDC80, FJX1 , TEAD4, S1 PR1 , WWC1 , NUAK2.

[0235] The different models were applied on below datasets to compare. All models were calibrated to provide a score between 0 and 100 where 100 represent maximum Hippo pathway activity and 0 represents no detectable Hippo pathway activity, all models are able to distinguish between active and inactive Hippo pathway activities in the selected proof points.

[0236] The below proofpoints were obtained from in house cell culture (proofpoint numbers 1 , 6, 7, 9, 23) public dataset GSE229066 (proofpoint numbers 2-5, 12, 16, 29, 30) public dataset GSE229071 (proofpoint numbers 8, 13), public dataset GSE262853 (proofpoint numbers 10, 15, 19, 21 , 22, 27), public dataset GSE215114 (proofpoint numbers 11 , 17, 20), public dataset GSE203421 (proofpoint number 14), public dataset GSE246578 (proofpoint numbers 18, 26), public dataset GSE186177 (proofpoint number 24), public dataset GSE231947 (proofpoint number 25), public dataset GSE197468 (proofpoint number 28).

[0237] The public datasets are described in the following publications:

[0238] GSE229066, GSE229071 : Hagenbeek et al., Nature Cancer, 4(6), 812-828.

[0239] GSE262853: Akao et al., Molecular Cancer Therapeutics, OF1-OF11.

[0240] GSE215114, GSE203421 : Hu et al. ELife, 11 , e80210.

[0241] GSE246578, GSE246583: Paul et al., Nature Communications, 16(1), 1743.

[0242] GSE186177: Li et al. The EMBO Journal, 42(4), e112184.

[0243] GSE197468: Li et al., Cell Discovery, 8, 103.

[0244] GSE231947: Zhang et al. Current Medical Science, 44(1), 81-92.

[0245] The proofpoints describe the following conditions:

[0246] 1. Calu-1 lung cancer cells treated with or without 1 pM TEAD-inhibitor GNE-7883 for 44 hours.

[0247] 2. OVCAR-8 ovarian cancer cells treated with or without TEAD-inhibitor GNE- 7883 for 24 hours.

[0248] 3. HCC1576 adrenal gland cells treated with or without TEAD-inhibitor GNE-7883 for 24 hours.

[0249] 4. HCC1576 adrenal gland cells treated with or without putative TEAD-inhibitor compound 3 for 24 hours.

[0250] 5. OVCAR-8 ovarian cancer cells treated with or without putative TEAD-inhibitor compound 3 for 24 hours.

[0251] 6. Calu-1 lung cancer cells treated with or without 0.1 pM YAP / TAZ-TEAD inhibitor IAG933 for 44 hours

[0252] 7. OVCAR-3 ovarian cancer cells treated with or without 1 pM TEAD-inhibitor GNE-7883 for 44 hours.

[0253] 8. NCI-H358 sotorasib-resistant KRAS G12C mutant lung cell line vs. sensitive cells, treated with AMG510 (Sotorasib, KRAS G12C inhibitor).

[0254] 9. OVCAR-3 ovarian cancer cells treated with or without 0.1 pM YAP / TAZ-TEAD inhibitor IAG933 for 44 hours. 10. MSTO-211 H Mesothelioma (lung cancer) cell line (PSEN1-LATS1 fusion and 42 bp deletion in exon 5 of the LATS2) treated with or without 1 pM TEAD inhibitor K- 975 for 48 hours.

[0255] 11. NCI-H226 Mesothelioma (lung cancer) cell line treated with or without 1 pM TEAD-inhibitor K-975 for 24 hours.

[0256] 12. HCC1576 Adrenal gland cell line treated with or without Hippo pathway inhibitor “compound 2” for 24 hours.

[0257] 13. NCI-H23 sotorasib-resistant KRAS G12C mutant lung cell line vs. sensitive cells, treated with AMG510 (Sotorasib, KRAS G12C inhibitor).

[0258] 14. NCI-H226 Mesothelioma (lung cancer) cell line treated with or without 1 pM TEAD inhibitor TM2 for 24 hours.

[0259] 15. NCI-H2052 Mesothelioma (lung cancer) cell line treated with or without 1 pM TEAD inhibitor K-975 for 48 hours.

[0260] 16. OVCAR8 Ovarian cancer cell line treated with or without Hippo pathway inhibitor “compound 2” for 24 hours.

[0261] 17. NCI-H226 Mesothelioma (lung cancer) cell line treated with or without 1 pM TEAD1 inhibitor VT103 for 24 hours.

[0262] 18. NCI-H226 Mesothelioma (lung cancer) cell line; TEAD sensitive, treated with or without TEAD inhibitor GNE-7883.

[0263] 19. Y-MESO-27 Mesothelioma (lung cancer) cell line treated with or without 1 pM TEAD inhibitor K-975 for 48 hours.

[0264] 20. NCI-H226 Mesothelioma (lung cancer) cell line treated with or without 1 pM TEAD inhibitor TM2 for 24 hours.

[0265] 21. ACC-MESO-1 Mesothelioma (lung cancer) cell line treated with or without 1 pM TEAD inhibitor K-975 for 48 hours.

[0266] 22. MSTO-211 H-KR Mesothelioma (lung cancer) cell line; TEAD resistant, treated with or without 1 pM TEAD inhibitor K-975 for 48 hours.

[0267] 23. Calu-1 Lung cancer cell line treated with or without 1 pM MST1 / 2 inhibitor XMU- MP-1 for 44 hours.

[0268] 24. C4-2 Prostate cancer cell line treated with or without 3 pM MST1 / 2 inhibitor XMU-MP-1 for 6 hours.

[0269] 25. NALM6 Lymphocytic cell line treated with or without 0.6mmol / L verteporfin (YAP inhibitor) for 48 hours. 26. NCI-H226_CL3 Mesothelioma (lung cancer) cell line; TEAD resistant; treated with or without TEAD inhibitor GNE-7883.

[0270] 27. NCI-H2052-KR Mesothelioma (lung cancer) cell line; TEAD resistant, treated with or without 1 M TEAD inhibitor K-975 for 48 hours.

[0271] 28. 786-0 Renal cell line treated with or without 4 M MST 1 / 2 inhibitor XMU-MP-1 for 6 hours.

[0272] 29. HCC1576 Adrenal gland cell line treated with or without Hippo pathway inhibitor “compound 4” for 24 hours.

[0273] 30. OVCAR8 Ovarian cancer cell line treated with or without Hippo pathway inhibitor “compound 4” for 24 hours.

[0274] 31. A-TU-8902 pancreatic adenocarcinoma cell line silenced for the YAP / WWTR1 gene (shYAP / WWTR1) in a cohort of 6-8 weeks Nu / Nu nude female mice. Resistant to TEAD inhibitor GNE-7883 vs. sensitive (shNTR and shYAP / WWTR1).

[0275] Pathway activity was calculated using the models (1A, 1 B, 2A, 2B) described above on the 31 proofpoints using an arbitrary scale from 0 to 100, 0 representing the theoretical lowest possible activity and 100 the theoretical highest possible Hippo pathway activity. The pathway values obtained are plotted in the table below and in line with expectations. Notably the individual models perform best on the dataset on which they are calibrated as expected. In all proofpoints where a difference in pathway activity is to be expected all the models were clearly able to distinguish between active and inactive pathway activity. It is noted that compound 4 appears to have no effect on Hippo pathway activity.

[0276] Proofpoint Model No. Tissue Cell line Condition 1A 1B 2A 2B

[0277] 1 Lung Calu-1 control 70 67 68 69

[0278] 1 Lung Calu-1 IAG933 17 14 48 47

[0279] 2 Ovary OVCAR-8 control 55 48 71 74

[0280] 2 Ovary OVCAR-8 GNE-7883 17 13 35 37

[0281] 3 Adrenal HCC1576 control 54 47 71 74

[0282] 3 Adrenal HCC1576 GNE-7883 18 14 36 38

[0283] 4 Adrenal HCC1576 control 54 47 71 74 4 Adrenal HCC1576 compound 3 22 17 39 42

[0284] 5 Ovary OVCAR-8 control 55 48 71 74

[0285] 5 Ovary OVCAR-8 compound 3 22 17 40 43

[0286] 6 Lung Calu-1 control 70 67 68 69

[0287] 6 Lung Calu-1 GNE-7883 34 22 52 52

[0288] 7 Ovary OVCAR-3 control 34 33 62 67

[0289] 7 Ovary OVCAR-3 GNE-7883 23 20 20 22

[0290] 8 Lung NCI-H358 AMG510 16 13 42 40

[0291] 8 Lung NCI-H358-R AMG510 49 39 56 56

[0292] 9 Ovary OVCAR-3 control 34 33 62 67

[0293] 9 Ovary OVCAR-3 IAG933 27 25 27 35

[0294] 10 Lung MSTO-211 H control 54 45 62 60

[0295] 10 Lung MSTO-211 H K-975 26 21 49 44

[0296] 11 Lung NCI-H226 control 64 63 78 81

[0297] 11 Lung NCI-H226 K-975 38 38 65 67

[0298] 12 Adrenal HCC1576 control 54 47 71 74

[0299] 12 Adrenal HCC1576 compound 2 32 28 53 56

[0300] 13 Lung NCI-H23 AMG510 10 8 26 27

[0301] 13 Lung NCI-H23-R AMG510 30 21 49 46

[0302] 14 Lung NCI-H226 control 66 65 76 80

[0303] 14 Lung NCI-H226 TM2 46 44 60 64

[0304] 15 Lung NCI-H2502 control 59 52 63 69

[0305] 15 Lung NCI-H2502 K-975 39 27 51 56

[0306] 16 Ovary OVCAR-8 control 55 48 71 74

[0307] 16 Ovary OVCAR-8 compound 2 33 29 56 59

[0308] 17 Lung NCI-H226 control 64 63 78 81

[0309] 17 Lung NCI-H226 VT103 40 40 67 69

[0310] 18 Lung NCI-H226 control 45 43 63 69

[0311] 18 Lung NCI-H226 GNE-7883 27 18 53 53

[0312] 19 Lung Y-MESO-27 control 49 38 65 66

[0313] 19 Lung Y-MESO-27 K-975 22 17 55 56

[0314] 20 Lung NCI-H226 control 64 63 78 81

[0315] 20 Lung NCI-H226 TM2 41 42 67 69 21 Lung ACC- M ESO- 1 control 27 26 55 61

[0316] 21 Lung ACC- M ESO- 1 K-975 19 17 40 47

[0317] 22 Lung MSTO-211 H-KR control 71 69 80 76

[0318] 22 Lung MSTO-211 H-KR K-975 59 49 73 70

[0319] 23 Lung Calu-1 control 70 67 68 69

[0320] 23 Lung Calu-1 XMU-MP-1 60 56 66 65

[0321] 24 Lung C4-2 control 11 10 14 20

[0322] 24 Lung C4-2 XMU-MP-1 7 6 7 11

[0323] 25 Lymphocyte NALM6 control 12 12 9 18

[0324] 25 Lymphocyte NALM6 verteporfin 7 6 5 13

[0325] 26 Lung NCI-H226-CL3 control 48 45 68 73

[0326] 26 Lung NCI-H226-CL3 GNE-7883 45 41 65 71

[0327] 27 Lung NCI-H2052-KR control 59 52 63 69

[0328] 27 Lung NCI-H2052-KR K-975 56 48 62 68

[0329] 28 Kidney 786-0 control 36 34 56 60

[0330] 28 Kidney 786-0 XMU-MP-1 33 31 58 57

[0331] 29 Adrenal HCC1576 control 54 47 71 74

[0332] 29 Adrenal HCC1576 compound 4 54 47 71 74

[0333] 30 Ovary OVCAR-8 control 55 48 71 74

[0334] 30 Ovary OVCAR-8 compound 4 54 48 72 75

[0335] 31 Pancreas PA-TU-8902 control 15 12 33 29

[0336] 31 Pancreas PA-TU-8902 YAP KD 14 11 31 29

[0337] Example 5 - 6 gene models

[0338] Based on the above data of Example 4 data it was concluded that all four models (1 A, 1 B, 2A, 2B) are suitable for determining Hippo pathway activity but each is more effective in a subset of proofpoints more resembling the proofpoint on which it is calibrated. As there is a set of 10 genes used in each of the four models (ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, WWC1) it was next speculated that a model based on (a subset of) these 10 genes could be a suitable model for non cell specific Hippo pathway model. In order to test this 6 random selections of six target genes from the 10 overlapping genes ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, WWC1 were used for 6 pathway models as follows:

[0339] Name Genes

[0340] L1g6-1 CCN2, F3, CCN1 , AMOTL2, TEAD4, WWC1

[0341] L1g6-2 CCN2, F3, CCN1 , AMOTL2, TEAD4, WWC1

[0342] L1g6-3 CCN2, F3, CCN1 , AMOTL2, TEAD4, WWC1

[0343] L1g6-4 CCN2, F3, CCN1 , AMOTL2, TEAD4, WWC1

[0344] L1gb-5 ANKRD1 , CCND1 , AMOTL2, CCDC80, TEAD4, WWC1

[0345] L1gc-6ANKRD1 , CCN2, CCND1 , CNN1 , CCDC80, WWC1

[0346] The models were calibrated on different cancer cell lines and using different specific inhibitors in line with Example 4. The models were applied on the same 31 proofpoints described above; resulting pathway activities are plotted in the table below:

[0347] Proofpoint Model No. L1g6-1 L1g6-2 L1g6-3 L1g6-4 L1g6-5 L1g6-6

[0348] 1 72 66 67 72 57 71

[0349] 1 13 10 36 46 34 12

[0350] 2 69 64 75 82 71 56

[0351] 2 24 19 38 44 11 9

[0352] 3 68 64 75 82 73 56

[0353] 3 27 23 39 46 14 9

[0354] 4 68 64 75 82 73 56

[0355] 4 32 27 42 49 19 15

[0356] 5 69 64 75 82 71 56

[0357] 5 33 28 42 50 21 15

[0358] 6 72 66 67 72 57 71

[0359] 6 28 22 46 55 38 20

[0360] 7 42 40 63 72 59 39

[0361] 7 24 23 11 17 18 18

[0362] 8 23 18 34 38 21 9

[0363] 8 52 46 60 65 33 42 9 42 40 63 72 59 39

[0364] 9 30 29 24 35 27 24

[0365] 10 64 58 66 71 42 54

[0366] 10 32 27 49 56 26 15

[0367] 11 87 86 84 88 65 80

[0368] 11 56 53 67 75 56 49

[0369] 12 68 64 75 82 73 56

[0370] 12 45 40 51 61 46 34

[0371] 13 22 19 32 42 15 2

[0372] 13 51 44 53 56 27 20

[0373] 14 75 71 80 85 70 77

[0374] 14 56 52 63 72 54 52

[0375] 15 55 48 66 75 53 33

[0376] 15 34 30 45 56 42 17

[0377] 16 69 64 75 82 71 56

[0378] 16 47 42 53 63 52 37

[0379] 17 87 86 84 88 65 80

[0380] 17 59 55 70 78 57 49

[0381] 18 52 47 70 78 54 50

[0382] 18 35 31 56 62 48 31

[0383] 19 53 48 61 67 41 39

[0384] 19 27 21 51 59 32 13

[0385] 20 87 86 84 88 65 80

[0386] 20 60 58 72 79 58 54

[0387] 21 43 37 57 67 28 35

[0388] 21 29 25 39 50 16 18

[0389] 22 78 75 81 85 75 74

[0390] 22 62 58 70 75 60 55

[0391] 23 72 66 67 72 57 71

[0392] 23 70 65 63 68 54 63

[0393] 24 19 19 15 20 28 17

[0394] 24 12 11 7 10 13 12

[0395] 25 11 11 10 16 10 0 25 8 7 8 13 7 0

[0396] 26 60 56 74 80 54 53

[0397] 26 57 52 71 78 52 49

[0398] 27 54 48 65 74 52 33

[0399] 27 54 48 64 74 52 33

[0400] 28 40 35 51 61 46 34

[0401] 28 54 49 63 70 43 48

[0402] 29 68 64 75 82 73 56

[0403] 29 68 64 74 82 73 56

[0404] 30 69 64 75 82 71 56

[0405] 30 69 65 76 83 75 58

[0406] 31 25 22 24 34 25 8

[0407] 31 24 22 24 33 21 5

[0408] CLAUSES

[0409] 1. A method for inferring the activity of the Hippo cellular signaling pathway in a sample, the method comprising: receiving the expression levels of three or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2, the expression levels of three or more Hippo target genes being determined in a sample; and inferring the Hippo cellular signaling pathway activity based on the three or more expression levels.

[0410] 2. The method according to clause 1 , wherein the inferring is based on evaluating a calibrated mathematical pathway model, preferably the calibrated mathematical pathway model is a probabilistic model, more preferably a Bayesian network model, or wherein the mathematical pathway model is based on one or more (pseudo-)linear combination(s) of the expression levels of the three or more Hippo target genes.

[0411] 3. The method according to clause 1 or 2, wherein the method further comprises a step of determining an activity level of a Hippo transcription factor (TF) element in the sample, the Hippo TF element controlling transcription of the three or more Hippo target genes, the determining being based on evaluating a calibrated mathematical pathway model relating the expression levels of the three or more Hippo target genes to the activity level of the Hippo TF element, and inferring the activity of the Hippo cellular signaling pathway in the sample based on the determined activity level of the Hippo TF element in the sample.

[0412] 4. The method according to any one of the preceding clauses, wherein the sample is obtained from a subject.

[0413] 5. The method according to any one of the preceding clauses, further comprising determining whether the Hippo cellular signaling pathway is operating at an undesired activity level in the sample based on the inferred activity of the Hippo cellular signaling pathway in the sample.

[0414] 6. The method of clause 5, further comprising recommending prescribing a drug for a subject that corrects for undesired activity level operation of the Hippo cellular signaling pathway, wherein the recommending is performed if the Hippo cellular signaling pathway is determined to be operating at an undesired activity level in the subject based on the inferred activity of the Hippo cellular signaling pathway.

[0415] 7. The method of clause 5 or 6, wherein the undesired activity level is a too low activity level or a too high activity level.

[0416] 8. The method of any one of clauses 5 to 7, wherein the undesired activity level of the Hippo cellular signaling pathway is an activity level in which the Hippo cellular signaling pathway operates as a promoter of, or fails to inhibit, or is an indication of one or more of the following disorders: cancer, an eye disease, cardiac disease, pulmonary disease, renal disease, a central nervous system disorder, a hepatic disease, or an immune dysfunction.

[0417] 9. The method according to any one of the preceding clauses, wherein the method further comprises a step of providing a treatment recommendation.

[0418] 10. The method according to any one of the preceding clauses, wherein the inferred Hippo cellular signaling pathway activity is used to predict an outcome for a subject.

[0419] 11. The method according to any one of the preceding clauses, wherein the inferred Hippo cellular signaling pathway activity is used to predict a treatment response for a subject.

[0420] 12. The method according to any one of the preceding clauses, wherein the method is a computer implanted method. 13. The method according to any one of clauses 1 to 11 , wherein the method involves a step of determining the three or more expression levels in the sample.

[0421] 14. A method for determining or predicting resistance of a tumor to a KRAS inhibitor, the method comprising: receiving the expression levels of three or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2, the expression levels of three or more Hippo target genes being determined in a sample obtained from the tumor; inferring the Hippo cellular signaling pathway activity based on the three or more expression levels, and predicting or determining the tumor to be or become resistant to KRAS inhibitors if the inferred Hippo cellular signaling pathway activity is low.

[0422] 15. The method according to clause 14, wherein the predicting or determining is done prior to treatment onset or after onset of treatment.

[0423] 16. The method according to clause 14 or 15, wherein the method comprises recommending a treatment.

[0424] 17. The method according to clause 16, wherein the recommended treatment is a KRAS inhibitor if the inferred Hippo cellular signaling pathway activity is high.

[0425] 18. The method according to clause 16, wherein the recommended treatment is an alternative treatment if the inferred Hippo cellular signaling pathway activity is low.

[0426] 19. The method according to clause 18, wherein the alternative treatment is selected from chemotherapy, surgery, immunotherapy, radiation therapy, or a combination of a KRAS inhibitor and a YAP / TAZ inhibitor.

[0427] 20. The method according to any one of clauses 14 to 19, wherein if resistance to a KRAS inhibitor is predicted or determined in the tumor the recommended treatment is a combination of a KRAS inhibitor and a YAP / TAZ inhibitor.

[0428] 21. The method according to any one of clauses 14 to 20, wherein the KRAS inhibitor is selected from Sotorasib (AMG 510), Adagrasib (MRTX849), GDC-6036, LY3499446, JNJ-74699157, D-1553, TNO155, JAB-3068, RMC-4630, RLY-1971 , and BBP-398, preferably sotorasib.

[0429] 22. The method according to any one of clauses 14 to 21 , wherein the tumor is bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, ependymoma, NF2-related Schwannomas, mesothelioma, and uveal melanoma, preferably selected from breast cancer, lung cancer, liver cancer, colorectal cancer, ependymoma, NF2-related Schwannomas, mesothelioma, and uveal melanoma.

[0430] 23. The method according to any one of clauses 14 to 22, wherein the tumor has a KRAS mutation.

[0431] 24. The method according to clauses 23 wherein the KRAS mutation is a mutation of the Glycine at position 12 of human KRAS, preferably wherein the mutation is a G12C, G12D or G12V mutation.

[0432] 25. The method according to any one of clauses 14 to 24, wherein the Hippo cellular signaling is determined according to the method as defined in any one of clauses 1 to 13.

[0433] 26. The method according to any one of clauses 14 to 25, wherein the method is used to predict if the tumor of a subject will become resistant to a KRAS inhibitor prior or during treatment with a KRAS inhibitor, is used to determine if the tumor of a subject is resistant to a KRAS inhibitor during treatment with a KRAS inhibitor, or to monitor if the tumor of a subject is or may become resistant to a KRAS inhibitor during treatment with a KRAS inhibitor.

[0434] 27. The method according to any one of clauses 14 to 26, wherein the prediction is further based on determining one or more of the MAPK, PI3K and NFkB cellular signaling pathway activities.

[0435] 28. The method according to clause 27, wherein a high MAPK, PI3K and / or NFkB cellular signaling pathway activity corresponds to a tumor being resistant to KRAS inhibitors and wherein a low MAPK, PI3K and / or NFkB cellular signaling pathway activity corresponds to a tumor being sensitive to KRAS inhibitors.

[0436] 29. A KRAS inhibitor for use in the treatment, prevention or amelioration of cancer in a subject, the use comprising receiving the expression levels of three or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2, the expression levels of three or more Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the three or more expression levels, and administering the KRAS inhibitor when the Hippo pathway is active.

[0437] 30. A YAP / TAZ inhibitor for use in the treatment, prevention or amelioration of cancer in a subject, the use comprising receiving the expression levels of three or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2, the expression levels of three or more Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the three or more expression levels, and administering the YAP / TAZ inhibitor when the Hippo pathway is inactive.

[0438] 31. A Hippo activator for use in the treatment, prevention or amelioration of cancer in a subject, the use comprising receiving the expression levels of three or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2, the expression levels of three or more Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the three or more expression levels, and administering the Hippo activator when the Hippo pathway is inactive.

[0439] 32. A method of treating, preventing or ameliorating cancer in a subject in need thereof, the method comprising: receiving the expression levels of three or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2, the expression levels of three or more Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the three or more expression levels, and if the Hippo pathway is active administering a KRAS inhibitor to the subject; or if the Hippo pathway is inactive administering a Hippo activator optionally combined with a KRAS inhibitor, a YAP / TAZ inhibitor optionally combined with a KRAS inhibitor or an alternative therapy selected from chemotherapy, surgery, immunotherapy, radiation therapy, a combination therapy of a KRAS inhibitor and a YAP / TAZ inhibitor, or a combination thereof.

[0440] 33. A kit of parts comprising polymerase chain reaction primers and optionally probes for specifically determining the expression levels of three or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2.

[0441] 34. Use of the kit according to clause 33 in inferring the Hippo cellular signaling pathway activity, the use comprising determining the expression levels of three or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2 using the polymerase chain reaction primers and optionally probes provided in the kit, and inferring the Hippo cellular signaling pathway based on the expression levels of the three or more Hippo target genes.

Claims

CLAIMS1. A method for inferring the activity of the Hippo cellular signaling pathway in a sample, the method comprising: receiving the expression levels of six or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NLIAK2, the expression levels of three or more Hippo target genes being determined in a sample; and inferring the Hippo cellular signaling pathway activity based on the three or more expression levels.

2. The method according to claim 1 , wherein the received expression levels comprise or consist of six or more Hippo target genes selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1.

3. The method according to claim 1 or 2, wherein the inferring is based on evaluating a calibrated mathematical pathway model, preferably the calibrated mathematical pathway model is a probabilistic model, more preferably a Bayesian network model, or wherein the mathematical pathway model is based on one or more (pseudo-)linear combination(s) of the expression levels of the six or more Hippo target genes, preferably . wherein the method further comprises a step of determining an activity level of a Hippo transcription factor (TF) element in the sample, the Hippo TF element controlling transcription of the six or more Hippo target genes, the determining being based on evaluating a calibrated mathematical pathway model relating the expression levels of the six or more Hippo target genes to the activity level of the Hippo TF element, and inferring the activity of the Hippo cellular signaling pathway in the sample based on the determined activity level of the Hippo TF element in the sample.

4. The method according to any one of the preceding claims, further comprising determining whether the Hippo cellular signaling pathway is operating at an undesired activity level in the sample based on the inferred activity of the Hippo cellular signalingpathway in the sample, preferably further comprising recommending prescribing a drug for a subject that corrects for undesired activity level operation of the Hippo cellular signaling pathway, wherein the recommending is performed if the Hippo cellular signaling pathway is determined to be operating at an undesired activity level in the subject based on the inferred activity of the Hippo cellular signaling pathway, more preferably wherein the undesired activity level is a too low activity level or a too high activity level, even more preferably wherein the undesired activity level of the Hippo cellular signaling pathway is an activity level in which the Hippo cellular signaling pathway operates as a promoter of, or fails to inhibit, or is an indication of one or more of the following disorders: cancer, an eye disease, cardiac disease, pulmonary disease, renal disease, a central nervous system disorder, a hepatic disease, or an immune dysfunction.

5. The method according to any one of the preceding claims, wherein the method further comprises a step of providing a treatment recommendation, and I or wherein the inferred Hippo cellular signaling pathway activity is used to predict an outcome for a subject, and I or wherein the inferred Hippo cellular signaling pathway activity is used to predict a treatment response for a subject.

6. The method according to any one of the preceding claims, wherein the method is a computer implanted method, or wherein the method involves a step of determining the six or more expression levels in the sample.

7. A method for determining or predicting resistance of a tumor to a KRAS inhibitor, the method comprising: receiving the expression levels of six or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2, preferably selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1 , the expression levels of six or more Hippo target genes being determined in a sample obtained from the tumor; inferring the Hippo cellular signaling pathway activity based on the six or more expression levels, andpredicting or determining the tumor to be or become resistant to KRAS inhibitors if the inferred Hippo cellular signaling pathway activity is low, preferably wherein the predicting or determining is done prior to treatment onset or after onset of treatment, more preferably wherein the method comprises recommending a treatment, even more preferably wherein the recommended treatment is a KRAS inhibitor if the inferred Hippo cellular signaling pathway activity is high, and / or wherein the recommended treatment is an alternative treatment if the inferred Hippo cellular signaling pathway activity is low, preferably wherein the alternative treatment is selected from chemotherapy, surgery, immunotherapy, radiation therapy, or a combination of a KRAS inhibitor and a YAP / TAZ inhibitor.

8. The method according to claim 7, wherein if resistance to a KRAS inhibitor is predicted or determined in the tumor the recommended treatment is a combination of a KRAS inhibitor and a YAP / TAZ inhibitor, preferably wherein the KRAS inhibitor is selected from Sotorasib (AMG 510), Adagrasib (MRTX849), GDC-6036, LY3499446, JNJ-74699157, D-1553, TNO155, JAB-3068, RMC-4630, RLY-1971 , and BBP-398, preferably sotorasib, more preferably wherein the tumor is bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, ependymoma, NF2-related Schwannomas, mesothelioma, and uveal melanoma, preferably selected from breast cancer, lung cancer, liver cancer, colorectal cancer, ependymoma, NF2-related Schwannomas, mesothelioma, and uveal melanoma.

9. The method according to claim 7 or 8, wherein the tumor has a KRAS mutation, preferably wherein the KRAS mutation is a mutation of the Glycine at position 12 of human KRAS, preferably wherein the mutation is a G12C, G12D or G12V mutation.

10. The method according to any one of claims 7 to 9, wherein the Hippo cellular signaling is determined according to the method as defined in any one of claims 1 to 6, preferably wherein the method is used to predict if the tumor of a subject will become resistant to a KRAS inhibitor prior or during treatment with a KRAS inhibitor, is used to determine if the tumor of a subject is resistant to a KRAS inhibitor during treatment with a KRAS inhibitor, or to monitor if the tumor of a subject is or may become resistant to a KRAS inhibitor during treatment with a KRAS inhibitor, more preferably wherein the prediction is further based on determining one or more of the MAPK, PI3K, WNT, and NFkB cellular signaling pathway activities, preferably wherein a high MAPK, PI3K, WNT, and / or NFkB cellular signaling pathway activity corresponds to a tumor being resistant to KRAS inhibitors and wherein a low MAPK, PI3K, WNT, and / or NFkB cellular signaling pathway activity corresponds to a tumor being sensitive to KRAS inhibitors.

11. A KRAS inhibitor for use in the treatment, prevention or amelioration of cancer in a subject, the use comprising receiving the expression levels of six or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2, preferably selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 , AMOTL2, CCDC80, FJX1 , TEAD4, and WWC1 , the expression levels of six or more Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the six or more expression levels, and administering the KRAS inhibitor when the Hippo pathway is active.

12. A YAP / TAZ inhibitor for use in the treatment, prevention or amelioration of cancer in a subject, the use comprising receiving the expression levels of six or more Hippo target genes selected from NPPB, ANKRD1 , IGFBP3, CCN2, F3, KISS1 , RASSF6, SNAPC1 , CCND1 , CCN1 , SKP2, AMOTL2, NT5E, DKK1 , ITGB2, CCDC80, FJX1 , AXL, NEXN, TEAD4, S1 PR1 , WWC1 , and NUAK2, preferably selected from ANKRD1 , CCN2, F3, CCND1 , CCN1 ,AMOTL2, CCDC80, FJX1, TEAD4, and WWC1, the expression levels of six or more Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the six or more expression levels, and administering the YAP / TAZ inhibitor when the Hippo pathway is inactive.

13. A Hippo activator for use in the treatment, prevention or amelioration of cancer in a subject, the use comprising receiving the expression levels of six or more Hippo target genes selected from NPPB, ANKRD1, IGFBP3, CCN2, F3, KISS1, RASSF6, SNAPC1, CCND1, CCN1, SKP2, AMOTL2, NT5E, DKK1, ITGB2, CCDC80, FJX1, AXL, NEXN, TEAD4, S1PR1, WWC1, and NUAK2, preferably selected from ANKRD1, CCN2, F3, CCND1, CCN1, AMOTL2, CCDC80, FJX1, TEAD4, and WWC1, the expression levels of six or more Hippo target genes being determined in a tumor sample obtained from the subject; inferring the Hippo cellular signaling pathway activity based on the six or more expression levels, and administering the Hippo activator when the Hippo pathway is inactive.

14. A kit of parts comprising polymerase chain reaction primers and optionally probes for specifically determining the expression levels of six or more Hippo target genes selected from NPPB, ANKRD1, IGFBP3, CCN2, F3, KISS1, RASSF6, SNAPC1, CCND1, CCN1, SKP2, AMOTL2, NT5E, DKK1, ITGB2, CCDC80, FJX1, AXL, NEXN, TEAD4, S1PR1, WWC1, and NUAK2, preferably selected from ANKRD1, CCN2, F3, CCND1, CCN1, AMOTL2, CCDC80, FJX1, TEAD4, and WWC1.

15. Use of the kit according to claim 14 in inferring the Hippo cellular signaling pathway activity, the use comprising determining the expression levels of six or more Hippo target genes selected from NPPB, ANKRD1, IGFBP3, CCN2, F3, KISS1, RASSF6, SNAPC1, CCND1, CCN1, SKP2, AMOTL2, NT5E, DKK1, ITGB2, CCDC80, FJX1, AXL, NEXN, TEAD4, S1PR1, WWC1, preferably selected from ANKRD1, CCN2, F3, CCND1, CCN1, AMOTL2, CCDC80, FJX1, TEAD4, and WWC1, and NUAK2 using the polymerase chain reaction primers and optionally probes provided in the kit, andinferring the Hippo cellular signaling pathway based on the expression levels of the six or more Hippo target genes.

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