WO2020165583A1 - Biomarqueurs - Google Patents

Biomarqueurs Download PDF

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Publication number
WO2020165583A1
WO2020165583A1 PCT/GB2020/050323 GB2020050323W WO2020165583A1 WO 2020165583 A1 WO2020165583 A1 WO 2020165583A1 GB 2020050323 W GB2020050323 W GB 2020050323W WO 2020165583 A1 WO2020165583 A1 WO 2020165583A1
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bcl
pah
sample
bmprii
patient
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PCT/GB2020/050323
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Talat Nasim
Hasnin CHOWDHURY
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University Of Bradford
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/321—Arterial hypertension
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse

Definitions

  • the present invention relates to new biomarkers for pulmonary arterial hypertension diagnosis and prognosis. Specifically, the invention relates to use of Bcl-xL and/or Bcl-xS or ratios therebetween as biomarkers for pulmonary arterial hypertension, the invention includes methods of diagnosis and prognosis of pulmonary arterial hypertension using the biomarkers and therapeutic interventions for pulmonary arterial hypertension.
  • Pulmonary Arterial Hypertension is a devastating and incurable cardiovascular disorder characterised by the remodelling of pre-capillary pulmonary arteries.
  • the pulmonary arteries are the arteries carrying blood from the right ventricle of the heart to the lungs for oxygenation.
  • the condition is characterised by abnormal proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs), endothelial cells (PAECs) and fibroblasts, leading to elevated pulmonary artery pressure, right-heart failure, and premature death.
  • PASMCs pulmonary arterial smooth muscle cells
  • PAECs endothelial cells
  • fibroblasts leading to elevated pulmonary artery pressure, right-heart failure, and premature death.
  • BMPR2 bone morphogenic protein type II receptor
  • BMP bone morphogenic protein
  • Bcl2 B-cell lymphoma 2
  • Bcl-x B-cell lymphoma X
  • Bcl-xL B-cell lymphoma X
  • SMAD-independent pathways such as p38MAPK and NF-KB are activated in experimental models of PAH, and that these pathways have been shown to enhance the expression of the Bcl-xL isoform, thereby exerting an anti-apoptotic phenotype.
  • apoptosis is greatly reduced in mouse PASMCs harbouring the pathogenic BMPR2 (p.R899X) mutation and the BMPRII deficiency potentiates the transforming growth factor b (TQRb) signalling pathway.
  • TQRb induces apoptosis in human B cells by transcriptional activation of Bcl-xL.
  • BMPRII deficiency elicits pro- proliferative and anti-apoptotic phenotype in PASMCs via the TGFb-associated kinase 1 (TAK1), but the underlying mechanisms by which apoptosis resistance is controlled in PAH remains unclear.
  • TGFb-associated kinase 1 TGFb-associated kinase 1
  • NT-proBNP B-type natriuretic peptide
  • At least one object of the invention is to provide biomarkers predictive for pulmonary arterial hypertension.
  • At least one object of the invention is to provide new therapeutic interventions for pulmonary arterial hypertension.
  • the inventors have discovered that the BMPRII-ALK1-Bcl-x pathway regulates cell-specific apoptosis in PAH. Whilst determination of the ratio of Bcl-xL and Bcl-xS transcripts offers the opportunity for a novel biomarker, targeting the BMPRII-Bcl-xL axis may provide a novel therapeutic intervention in PAH.
  • Bcl-x as a biomarker for PAH, wherein the Bcl-x is either Bcl-xL and Bcl-xS.
  • the biomarker is a DNA molecule or an RNA transcript or is a protein.
  • the biomarkers the present invention may be used in conjunction with a mutation in the BMPRII gene, wherein said mutation in the BMPRII gene indicates that the patient has or is at risk of developing PAH.
  • the biomarkers the present invention may be used in conjunction with a reduced level of either BMPR2 transcript or BMPRII protein, wherein the said deficiency indicates that the patient has or is at risk of developing PAH.
  • an in vitro method for diagnosis of PAH or pre-forms thereof in a subject comprising
  • an in vitro method of identifying a subject afflicted with, suspected to be afflicted with or having a risk of developing PAH comprising:
  • an in vitro method for monitoring the development, progress or relapse of PAH or pre-forms in a subject comprising:
  • step ii) comparing levels or amounts of Bcl-xL determined in step i) to the level or amount detected in step ii) or to a reference value or comparing ratios of levels or amounts of Bcl-xL to Bcl-xS determined in step i) to the level or amount detected in step ii) or to a reference value.
  • the diagnosis, monitoring the development, progress or relapse of PAH or pre forms in a subject afflicted with, suspected to be afflicted with or being at risk of developing PAH is based on any one or more of the following criteria:
  • the sample is selected from the group consisting of: a whole blood, sample, a serum sample, a saliva sample, a lymphocyte enriched blood sample, isolated lymphocyte cells, lung tissue, arterial tissue, isolated lymphocytes, isolated pulmonary arterial smooth muscle cells and isolated pulmonary arterial endothelial cells
  • the patient is human.
  • the said pre-determined reference level is the average level of Bcl-xL or Bcl-xS in a control patient or a set of control patients that do not suffer from or are likely to suffer from PAH.
  • the methods of the present invention further include the steps of determining if the patient has a mutation in the BMPRII gene, wherein the mutation in the BMPRII gene indicates that the patient has or is at risk of developing PAH.
  • biomarkers the present invention may be used in conjunction with a reduced level of either BMPR2 transcript or BMPRII protein, wherein the said deficiency indicates that the patient has or is at risk of developing PAH.
  • a method of treatment for PAH comprising administering any one or more of the following:
  • the modulator is selected from the group comprising small molecule, biologies (e.g. antibody and protein), DNA (e.g. oligos) and RNA molecules (e.g. RNA oligos, siRNAs).
  • small molecule e.g. antibody and protein
  • DNA e.g. oligos
  • RNA molecules e.g. RNA oligos, siRNAs.
  • any one or more of the group comprising a Bcl-xL inhibitor, a Bcl-xS agonist and a modulator that alters the ratio of Bcl-xL and BcL-Xs transcripts for the treatment of PAH.
  • the Bcl-xL inhibitor is 2, 3 DCPE hydrochloride or a biosimilar.
  • the Bcl-xL inhibitor is used to increase apoptosis in cells of the pulmonary artery.
  • the cells are pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells.
  • a Bcl-xL inhibitor for use in the treatment of PAH.
  • kits for diagnosing pulmonary arterial hypertension comprising a compound or agent for detecting levels of Bcl- xL and/or Bcl-xS, or the ratio of Bcl-xL to Bcl-xS in a sample isolate from a patient and instructions for using the kit.
  • kits for diagnosing pulmonary arterial hypertension comprising: i) a detectably labelled agent that specifically binds to Bcl-xL and/or Bcl-xS nucleic acid or a detectably labelled agent that specifically binds to Bcl-xL and/or Bcl-xS polypeptide and ii) reagents for performing a diagnostic assay.
  • an assay device for testing if a patent has pulmonary arterial hypertension comprising a compound or agent for detecting levels of Bcl-xL and/or Bcl-xS, or the ratio of Bcl-xL to Bcl-xS in a sample isolate from a patient.
  • FIG. 1A Alternative splicing of the Bcl-x gene. Selection of the upstream 5’ splice site (5’ss) generates the anti-apoptotic Bcl-xL long isoform, while the downstream 5’ splice site produces the pro-apoptotic short isoform, Bcl-xS.
  • Figure 1 BB Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of Bcl-xS and Bcl-xL transcripts derived from circulating leukocytes of PAH patients with and without BMPR2 mutations.
  • RT-PCR Reverse transcriptase polymerase chain reaction
  • Controls 1-6 are RNAs derived from healthy subjects whilst Control 7 is commercially available RNA (Clontech).
  • Figure 1 C Quantification of the RT-PCR image.
  • Figure 1 D RT-PCR analysis of Bcl-x transcripts in human lung tissue from a PAH subject harbouring a BMPR2 mutation (p.835X) who underwent combined heart and lung transplantation. The control lung RNAs are from a commercially available source (Clontech).
  • Figure 1 E Quantification of RT-PCR by image analysis. The mean densities of bands derived from three independent experiments are shown. **P ⁇ 0.01 , ***P ⁇ 0.001 compared with control as indicated.
  • FIG. 3A Determination of the alternative splicing of Bcl-x pre-mRNA in human PASMCs. RT-PCR analysis of Bcl-x transcripts in human PASMCs from a PAH subject harbouring the BMPR2 mutation (p.R899X).
  • the hTERT-PAH-PASMCs are the p.R899X-PAH PASMCs which were immortalized with the expression of catalytic subunit of human telomerase (hTERT) gene (Nasim et al, unpublished data).
  • Figure 3C Diagram depicting the roles of Bcl-xL and Bcl-xS isoforms in apoptosis.
  • Figure 3D effect of BMPR2 mutation on apoptosis in mouse PASMCs as determined by caspase 3/7 activities.
  • Apoptosis was induced by the established chemical staurosporine (ST, 1.25mM).
  • FIG. 3F Diagram depicting the role 2,3 DCPE hydrochloride (TOCRIS), which selectively inhibits the activity of Bcl-xL isoforms on apoptosis.
  • FIG. 4A Either BMPRII or ALK1 down-regulates the expression of Bcl-xL transcripts in PAECs.
  • Figure 4C Effects of siRNA knock-down on BMPR2 and ( Figure 4D) A/.K ⁇ .
  • FIG. 4E RT-PCR analysis of Bcl-x transcripts in HEK293T cells either transfected with a construct containing the BMPR2 gene or stimulated with BMP9 (10ng/ml) ligand.
  • Figure 4G RT-PCR analysis of Bcl-xS and Bcl-xL transcripts in PAECs stimulated with BMP9 ligand (10ng/ml) ( Figure 4H).
  • Figure 4J Diagram of the BMPRII domain structure depicting the ligand binding (LB), transmembrane (TM), kinase (KD) and C-terminal extension (CTD). The numbers below indicating relative amino acids, whilst the mutations tested are indicated above.
  • HEK293T cells were transfected either with the wild-type or mutant BMPRII receptors, ALK1 or ALK3 receptor and the relative cell viability was determined. The value derived from untreated control cells was set as 100. ***P ⁇ 0.001 compared with mock transfected cells, NS-non significant. Data are presented as mean ⁇ SEM from 3-6 independent experiments.
  • FIG. 5A Diagram depicting the effect of BMP signalling cascade on caspase 3/7 activities.
  • Figure 5C Diagram depicting the BMP signalling cascade as determined by the BMP responsive 3GC2-Lux reporter assay.
  • the luc-gal activities of cells at Oh time point were set as 100. **P ⁇ 0.01 , ***P ⁇ 0.001 compared with control treated for 0 hour.
  • HEK293T cells transfected with either BMPRII, ALK1 and ALK3 receptors alone or BMPRII in combination with ALK1 and ALK3 receptors.
  • Apoptosis was induced by treating the cells with staurosporine (1.25mM) for 16 hours (Figure 5G).
  • Data are presented as mean ⁇ SEM from 3-6 independent experiments.
  • Figure 5H ALK1 and BMPRII significantly inhibit staurosporine (ST)-induced caspase activity.
  • ALK1- BMPRII-mediated reporter activation was significantly increased compared with cells stimulated with either of BMP4 or BMP9 ligands.
  • HEK293T cells transfected with the reporter plasmids together with BMPRII and stimulated with either BMP4 (10ng/ml) or BMP9 (10ng/ml) ligands for overnight (n 8).
  • cells were co-transfected in combination with either wild-type or mutant BMPR-II receptor together with ALK1-DBD and pTN 114 reporter.
  • Model depicting the regulation of apoptosis resistance in PAH Model depicting the regulation of apoptosis resistance in PAH.
  • PASMCs either BMPR2 mutations or BMPRII receptor deficiency potentiate the expression of Bcl-xL transcripts leading to apoptosis resistance.
  • the receptor deficiency reduces the Bcl-xL expression leading to anti-survival effects.
  • the pro-survival phenotype of PAECs is mediated via the BMPRII and ALK1 receptors.
  • ALK1 and ALK3 receptors work synergistically with the BMPRII and in the event of pathogenic BMPR2 mutations, the efficiency of interactions of BMPRII with type I receptors is greatly reduced which increases the susceptibility of PASMCs and PAECs to undergo impaired apoptotic pathway but not sufficient to trigger PAH. The presence of an additional stimulus may further exacerbate the apoptosis-resistance process leading to vascular remodelling.
  • the Bcl-x gene may represent a potential biomarker and druggable target for PAH. Straight arrows indicate activation; T-shapped line indicates inhibition.
  • vascular remodelling observed in PAH lungs is caused by abnormal apoptosis of PASMCs and PAECs, but the underlying mechanisms by which apoptosis is controlled in PAH have remained elusive.
  • the inventors report a novel mechanism which demonstrates that the dysregulated apoptosis in PAH is controlled via the BMPRII-Bcl-x axis. It is demonstrated herein a number of ways that BMPR2 haploinsufficiency modulates the preferential expression of the anti-apoptotic Bcl-xL over the pro-apoptotic Bcl-xS transcripts both in PAH patients and in an animal model.
  • Bcl-2 family have been identified as biomarkers for PAH, providing early detection of a difficult to diagnose disease.
  • Bcl-xL and Bcl-xS have been identified as especially relevant to the diagnosis of PAH.
  • the involvement of anti-apoptotic Bcl-xL protein in developing apoptosis resistance in many cancers is well documented, but the underlying mechanisms by which preferential expression of the Bcl-x transcripts is regulated in PAH is not known.
  • a member of this family of genes is B-cell lymphoma X (Bcl-x), which generates both pro- (Bcl-xS) and anti-apoptotic (Bcl-xL) isoforms via alternative splicing
  • the Bcl-2 family consists of a number of evolutionarily-conserved proteins that share Bcl-2 homology (BH) domains.
  • the Bcl-2 family is most notable for their regulation of apoptosis, a form of programmed cell death, at the mitochondrion.
  • the Bcl-2 family proteins consists of members that either promote or inhibit apoptosis, and control apoptosis by governing mitochondrial outer membrane permeabilization (MOMP), which is a key step in the intrinsic pathway of apoptosis.
  • MOMP mitochondrial outer membrane permeabilization
  • Bcl-x is used to refer to both Bcl-x polypeptides, transcripts and nucleic acid molecules. Preferably the polypeptides, transcripts or nucleic acid molecules are human. “Bcl-x” is short form for Bcl-2 family proteins, for example Bcl-xL and Bcl-xS (also referred to as Bcl-x(L) and Bcl-x(S) in the literature and herein). All proteins belonging to the Bcl-2 family contain either a BH1 , BH2, BH3 or BH4 domain.
  • All anti-apoptotic proteins contain BH1 and BH2 domains, some of them contain an additional N-terminal BH4 domain (Bcl-2, Bcl-x(L) and Bcl-w), which is also seen in some pro-apoptotic proteins like Bcl-x(S).
  • the BH3 domain is also present in some anti-apoptotic protein, such as Bcl-2 or Bcl-x(L).
  • the three functionally important Bcl-2 homology regions (BH1 , BH2 and BH3) are in close spatial proximity.
  • Bcl-x(L) The long form (Bcl-x(L), displays cell death repressor activity, but the short isoform (Bcl-x(S)) and the b-isoform (Bcl-cb) promote cell death.
  • Bcl-x(L), Bcl-x(S) and Bcl-cb are three isoforms derived by alternative RNA splicing.
  • Bcl-2-like 1 or BCL2L1 is a human gene. Through alternative splicing, it encodes both of the human proteins Bcl-xL and Bcl-xS.
  • Bcl-x(L) is 233 amino acidyl residues (aa) long and exhibits a single very hydrophobic putative transmembrane a-helical segment (residues 210-226) when in the membrane.
  • the Bcl-xL and Bcl-xS nucleic acid molecules comprises the nucleotide sequences or polypeptides known in the art, or a fragment of any of these nucleotide sequences or polypeptides.
  • the Bcl-xL and Bcl-xS nucleic acid molecules or polypeptides comprise or consist of a nucleotide sequence or polypeptide which is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the entire length of the nucleotide sequences known in the art.
  • the Bcl-xL and Bcl-xS nucleic acid molecules may comprises or consists of a nucleic acid sequence that encodes the Bcl-xL and Bcl-xS polypeptide, as known in the art.
  • the Bcl-xL and Bcl-xS nucleic acid molecules comprises or consists of a nucleic acid sequence that encode an allelic variants of the Bcl-xL and Bcl-xS polypeptides, or preferably functional allelic variants.
  • polypeptides may have "non-critical” amino acid residues that can be altered from the wild-type sequence or "conservative amino acid substitutions” wherein amino acid residues are replaced with amino acid residues having a similar side chains, as is well known in the art.
  • amino acid residues are replaced with amino acid residues having a similar side chains, as is well known in the art.
  • homology and “identity” are used interchangeably. Calculations of sequence homology or identity between sequences are performed with methods and techniques standard in the art.
  • biomarker refers to a naturally occurring biological moiety or characteristic by which a particular pathological or physiological process or disease can be identified. Generally, diagnosis will take place in vitro.
  • ratio is the quantitative relation between two amounts showing the number of times one value contains or is contained within the other.
  • the various combinations included within the scope of the present inclusion include Bcl-xL vs Bcl-xS, Bcl-xL vs BMPR2, Bcl-xL vs GAPDH or an internal house-keeping gene, Bcl-xS vs BMPR2
  • a“a sample isolated from the patient” or a“biological sample” or a“patient sample” refers to tissues, cells and biological fluids isolated from a patient, as well as tissues, cells and fluids present within a patient.
  • the sample may be a urine sample, a blood sample, a serum sample, a sputum sample, a faecal sample, a biopsy of body tissues, for example a biopsy of transplanted lung tissue, a cerebro-spinal fluid sample, a semen sample or a smear sample.
  • Preferred samples are a whole blood sample, a serum sample, a leukocyte enriched blood sample, isolated (circulating) leukocyte cells, a lung tissue sample, an arterial tissue sample, a pulmonary arterial tissue sample, isolated leukocyte, isolated pulmonary arterial smooth muscle cells, isolated fibroblasts and isolated pulmonary arterial endothelial cells.
  • the sample is a whole blood sample.
  • the sample is a lymphocyte sample.
  • the sample is any biological sample containing target molecules by which Bcl-xL and/or Bcl-xS can be detected.
  • target molecules are biological molecules, suitable target molecules may be selected from: proteins, or precursors, or variants produced on translation of the transcripts produced when the gene is expressed; and nucleic acids (for example DNA or RNA) encoding said proteins.
  • patient refers to an individual, e.g., a human, having or at risk for having pulmonary arterial hypertension (PAH). The patient may be at risk from PAH or may be part of an at risk patient population for PAH (e.g. age related, smoker, weight related) or suspected to be part of an increased risk population.
  • PAH pulmonary arterial hypertension
  • the patient may have a genetic predisposition or risk of developing PAH.
  • the patient may have a heterozygous mutation of bone morphogenetic protein type-ll receptor (BMPR2 ), and as such be at risk of developing PAH.
  • BMPR2 bone morphogenetic protein type-ll receptor
  • the patient may be exhibiting other symptoms of PAH, such as increased fatigue and reduced exercise capacity.
  • the patient may be demonstrating other known early stage symptoms of PAH, or show signs from scans such as those described above.
  • Reference herein to“pre-form” is intended to include any early stage symptoms or pathological changes associated with PAH and typically prior to full blown PAH.
  • nucleic acid molecule includes DNA molecules (e.g. a cDNA or genomic DNA) and RNA molecules (e.g. a mRNA) and analogs of the DNA or RNA generated, e.g. by the use of nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • the level of expression of the Bcl-xL and Bcl-xS transcripts can be measured in a number of ways, including: measuring the mRNA encoded by the Bcl-xL and Bcl-xS nucleic acid molecule; measuring the amount of polypeptide encoded by the Bcl-xL and Bcl-xS nucleic acid molecule; or measuring the activity of the polypeptide encoded by the Bcl-xL and Bcl-xS nucleic acid molecule. Any known in the art method of measuring mRNA can be utilised, e.g. Southern or Northern blot analysis, polymerase chain reaction or probe arrays.
  • the level of Bcl-xL and Bcl-xS mRNA in a sample may be evaluated with nucleic acid amplification, for example by RT-PCR and QPCR, ligase chain reaction, self-sustained sequence replication, transcriptional amplification or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. Any known protein detection methods may be used to measure the level of activity of the polypeptide encoded by the Bcl-xL and Bcl-xS nucleic acid molecule in a sample.
  • protein detection methods comprise contacting an agent that selectively binds to a polypeptide, for example an antibody, preferably, the agent or antibody is labeled, for example with a detectable label.
  • a polypeptide for example an antibody
  • Suitable antibodies may be polyclonal or monoclonal.
  • An antibody fragment such as a Fab or F(ab')2 may be used.
  • labeled refers to direct labeling of the probe or antibody by coupling (i.e. physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance.
  • the level of polypeptide in a sample may be determined by techniques known in the art, such as enzyme linked immunosorbent assays (ELISAs), immunoprecipitation, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis.
  • ELISAs enzyme linked immunosorbent assays
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • Western blot analysis for in vivo detection of a polypeptide, a labeled antibody may be introduced into a patient. Such an antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the methods may comprise comparing the level or activity of Bcl-xL and Bcl-xS in a patient sample with the level or activity of Bcl-xL and Bcl-xS in a control sample or with a predetermined reference level for Bcl-xL and Bcl-xS.
  • Mass spectrometry methods could also be used to detect Bcl-xL and/or Bcl-xS levels.
  • control sample refers to a sample having a normal level of Bcl-xL and Bcl-xS expression, for example a sample from a healthy subject not having or suspected of having PAH, or alternatively a sample from the same subject that the biological test sample is obtained from, for example a sample obtained prior to having or being suspected of having PAH or prior to PAH disease progression, or a sample taken earlier in time.
  • a“per-determined reference level” or“reference level” may comprise Bcl-xL and/or Bcl-xS expression level from a reference database, which may be used to generate a pre-determined cut off value, i.e.
  • a diagnostic score that is statistically predictive of a symptom or disease or lack thereof or may be a pre-determined reference level based on a standard population sample, or alternatively, a pre-determined reference level based on a subject's base line level of expression, i.e. prior to having or being suspected of having PAH or prior to PAH disease progression.
  • diagnosis may be based on a normalized expression level Bcl-xL and/or Bcl-xS (transcript) expression level, normalized by correcting the absolute expression level in a sample by comparing its expression to the expression of a reference that is not a marker, e.g. a mRNA, such as an mRNA that is constitutively expressed (e.g. GAPDH).
  • a marker e.g. a mRNA, such as an mRNA that is constitutively expressed (e.g. GAPDH).
  • This normalization allows the comparison of the expression level in one sample to another sample, or between samples from different sources.
  • This normalized expression can then optionally be compared to a reference level or control. This could be to another transcript or protein marker in the sample.
  • an assay device for example a solid support such as an array or a chip, that has attached to a surface thereof a compound or agent capable of detecting a Bcl-xL and/or Bcl-xS polypeptide or nucleic acid.
  • compound or agent capable of detecting a Bcl-xL and/or Bcl-xS polypeptide is an anti- Bcl-xL and/or Bcl-xS antibody.
  • the assay device further comprises at least one additional compound or agent for detecting a further biomarker, preferably a biomarker for PAH.
  • kits for detecting the presence of Bcl-xL and/or Bcl-xS in a biological sample can include a compound or agent capable of detecting a Bcl-xL and/or Bcl-xS polypeptide or nucleic acid in a biological sample.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect Bcl-xL and/or Bcl-xS polypeptide or nucleic acid.
  • the kit can be a kit of diagnosing PAH in a patient, comprising: a detectably labelled agent that specifically binds to a Bcl-xL and/or Bcl-xS polypeptide or a detectably labelled agent that specifically binds to a Bcl-xL and/or Bcl-xS nucleic acid; and reagents for performing a diagnostic assay.
  • the agent may be an antibody or a nucleic acid molecule.
  • the kit can be an anti-body-based kit, as is well known in the art, with antibodies conjugated to detectable agents, or a oligonucleotide-based kit, including a nucleotide probe, e.g., a detectably labeled primer, which hybridizes to a nucleic acid molecule of interest or a pair of primers for amplifying the nucleic acid molecule of interest.
  • the kits can also include components necessary for detecting the detectable agent (e.g., an enzyme or a substrate).
  • the kits can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained.
  • the methods of diagnosis and prognosis described herein may further comprise a step of treating a patient or organ, on the basis of the diagnosis or prognosis.
  • the step of treating a patient or organ may, by way of example only, involve oxygen therapy, blood thinners, treatments with the aim of improving or maintaining heart function, calcium channel blockers, blood pressure medication, blood vessel enlarging therapies, atrial septostomy, a lung transplantation operation, or any other method of treating or ameliorating PAH or associated symptoms of PAH.
  • a“Bcl-xL inhibitor” is a synthetic or biological substance which blocks, retards, or prevents expression or generation of Bcl-xL.
  • the Bcl-xL inhibitor is 2, 3 DCPE hydrochloride or a biosimilar.
  • the inventors found preferential expression of the anti-apoptotic Bcl-xL transcripts in circulating leucocytes of PAH patients with and without BMPR2 mutations. Second, the increased expression of Bcl-xL transcripts was also observed in lungs of a PAH patient who had undergone combined heart and lung transplantation.
  • the BMPR2 mutations included in this study are nonsense, insertion, and deletion mutations, and hence transcripts harbouring these alleles are likely to be degraded through the nonsense-mediated decay (NMD) pathway, creating a state of haploinsufficiency.
  • NMD nonsense-mediated decay
  • the ratios of Bcl-xL to Bcl-xS and Bcl-xL to GAPDH were increased. Consistent with previous observations, elevated levels of Bcl-xL protein were also found in pulmonary artery of chronic hypoxia treated rats with vascular remodelling.
  • ERK1/2 activates the p90RSK, which potentiates the transcription factor CREB and induces the expression of Bcl-xL and Bcl-2 transcripts.
  • ECs derived from idiopathic PAH (I PAH) patients show elevated levels of phosphorylated ERK1/2 and Bcl-xL expression leading to apoptosis resistance.
  • TNF-a has been shown to activate both pro- apoptotic and anti-apoptotic pathways. Whilst it induces apoptosis by activating caspase-8 and -10, its anti-apoptotic activity is mediated via NF-KB, which induces the expression of anti- apoptotic Bcl-2 family of proteins.
  • the PI3K/AKT pathway inhibits the expression of Bcl-xL transcripts by inhibiting the activation of FoxO transcription factor (35,36).
  • BMPRII controls the expression of Bcl-xL transcripts.
  • the inventors showed that in PAH-PASMCs harbouring the pathogenic BMPR2 (p.R899X) mutation, the expression of anti-apoptotic Bcl-xL transcripts was greatly increased, indicating that BMPRII dysfunction might potentiate resistance to apoptosis.
  • siRNA knock-down of either the BMPR2 or ALK1 genes inhibited the expression of Bcl-xL in PAECs.
  • BMPRII receptor or BMP9 stimulation increased the expression of Bcl-xL in HEK293T cells.
  • BMPRII deficiency promotes the expression of Bcl-xL transcripts in PASMCs, whilst inhibits it in ECs.
  • the increased expression of Bcl-xL transcripts may contribute to differential apoptotic effects in these cells.
  • the inventors found that dysfunctional BMPRII signalling exerted anti-apoptotic phenotype in PASMCs whilst deficiency of this receptor led to apoptosis in PAECs.
  • BMPRII receptor overexpression of BMPRII receptor promoted cell survival and this effect was greatly reduced in the presence of pathogenic BMPR2 mutations.
  • the inventors provided extensive evidence that the pro-survival effect of BMPRII signalling was mediated through the ALK1 receptor and that these two receptors worked synergistically. Firstly, caspase activity was greatly reduced following co-expression of BMPRII with ALK1 but not with ALK3 receptor; activation of the BMP9/ALK1/BMPRII axis in endothelial cells showed anti-apoptotic phenotypes. Secondly, cells co-expressed with BMPRII and ALK1 , promoted BMP-responsive reporter activity and mutation either in BMPRII or in ALK1 impaired the reporter activation.
  • the ALK1 receptor works synergistically with the BMPRII and in the event of pathogenic BMPR2 mutations, the BMPRII-ALK1-mediated signalling is greatly reduced which increases the susceptibility of endothelial cells to undergo apoptosis but not sufficient to trigger PAH.
  • a critical reduction in BMPRII-mediated signalling or the presence of an additional stimulus may trigger apoptosis-resistance of PASMCs leading to vascular remodelling.
  • BMPRII deficiency impairs apoptosis by modulating the alternative splicing of Bcl-x transcripts, a finding observed in circulating leukocytes, lungs of PAH patients, hypoxic PAH rat lungs as well as in disease relevant human PASMCs and PAECs. Whilst BMPRII deficiency elicits anti-apoptotic effects in PASMCs, the receptor dysfunction induces pro-apoptotic responses in PAECs. The pro-survival effects of BMPRII are mediated through the ALK1 but not the ALK3 receptor in PAECs/HEK293T cells.
  • BMPRII interacts with the ALK1 receptor and in the event of pathogenic BMPR2 mutations, this interaction is greatly reduced resulting in impaired signalling events.
  • the inventors propose that assessing Bcl-x transcripts in patient samples offers the opportunity for developing a novel biomarker and that targeting the BMPRII-Bcl-xL axis may provide a novel therapeutic intervention in PAH.
  • Control samples are healthy individuals, all of which have no mutations in the BMPR2 gene.
  • Human lung tissues are from a PAH subject harbouring a BMPR2 mutation (p.835X) who underwent combined heart and lung transplantation.
  • the control human lung RNAs are from a commercially available source (Clontech).
  • PAH-PASMCs were isolated from the proximal pulmonary arterial vessel segment (5-8mm diameter) obtained from the lung sample of a HPAH patient with a pathogenic BMPR2 mutation (p.R899X) undergoing lung transplantation.
  • PASMCs of wild type ( bmpr2 +/+ ) and knock-in mice harbouring the PAH- associated bmpr2 nonsense mutation (p.R899X) were derived from explants.
  • the luminal surface of the pulmonary artery was cut to open and endothelium was gently scrapped off using scalpel blade.
  • the adjacent adventitia was stripped off and the medial explants were cut into 4-9 mm 2 sections. These segments were then plated into T25 cm 2 flasks and allowed to adhere.
  • DM EM containing 20% FBS and antibiotic (Penicillin/Streptomycin)- antimycotic (Amphotericin B) was added to the flasks after 2 hours (40,41). Cells were grown to confluency and used between passages 4-10. The phenotype of the isolated cells was confirmed by immunostaining using antibody to smooth muscle-specific a-actin (Sigma).
  • RNA isolation, cDNA synthesis and reverse Transcriptase PCR RT-PCR
  • RNAs were isolated from mammalian cells and circulating leukocytes using either TRI-Reagent (Sigma) or RNeasy Purification Kit (Qiagen). cDNAs were synthesized using random primers and MMLV Reverse Transcriptase (Promega) following manufacturer’s protocol. Total lung tissue RNA was extracted using the Trizol method (Invitrogen). 2 pg RNA was then reverse transcribed using Thermoscript RT-PCR kits (Invitrogen). The PCR was carried out using Hi- Fidelity Extensor Master Mix (ABgene).
  • Quantitative PCR for determining transcripts of bmpr2 , and b -2 microglobulin ⁇ -2mg were performed using TaqMan Gene Expression Assay (Applied Biosystems) on either 7900HT Fast Real-Time PCR system (Applied Biosystems) or Step One Plus (Applied Biosystems) according to the manufacturer’s protocol.
  • Apoptosis assays were carried out using Caspase-Glo ® 3/7 Assay (Promega) following manufacturer’s instruction. Luminescence was recorded using either an ORION-II or an ORION-L Plate Luminometer (Berthold) at 30 and 60 minutes intervals. The cell survival activity was determined using CellTiter-Glo Cell Viability Assay (Promega). Short interfering RNA (siRNA)-mediated knock-down of ALK ⁇ and BMPR2 genes siGENOME SMART pool siRNAs (Dharmacon) were used to knock down A/.K ⁇ and BMPR2 genes. The siGENOME SMART pool is a mixture of 4 siRNAs, all designed to target different regions of the single gene of interest. PAECs were transfected with siRNAs either in 96 well plate (1X10 4 ) or 6-well plate (1X10 6 ) using Dharmafect 1 (Dharmacon) following manufacturer’s protocol and incubated for 72 hours.
  • the coding sequence of human ALK1 (accession number BC017715) lacking the first amino acid was cloned into BamHI and Xbal sites of pTN 11 1 vector.
  • the resulting plasmid contains a N-terminal DNA binding domain (DBD) adjacent to the human ALK1 , the fidelity of which was verified by restriction analyses and sequencing.
  • DBD N-terminal DNA binding domain
  • Efficiency of protein-protein interactions between ALK1 and BMPRII in mammalian cells was determined as previously reported (http://www.natureprotocols.com/2007/06/28/efficiency of proteinprotein i l .php).
  • the intracellular domain of BMPR2 was fused with VP-16 activation domain whilst ALK1 was fused with a DBD.
  • Plasmids encoding both fusion proteins were transfected into HEK293T cells together with the pTN 114 dual reporter plasmid. In the event of an interaction both reporter proteins will be generated with the ratio of reporter activities producing a measure of the efficiency of protein-protein interactions.
  • the BMPR2 mutations were nonsense (p.L287X), frameshift (c.A796fsX6) and deletion (c.A497-503del, c.768+3delA), all of which are likely to introduce a premature termination codon into the reading frame.
  • a substantial reduction in the pro-apoptotic transcript, Bcl-xS, and an increase in the anti-apoptotic Bcl-xL isoform were observed in PAH cases compared to control subjects ( Figure 1 B-C).
  • the inventors next determined the relationship between defective BMPRII signalling and Bcl-x splicing in lung tissues of a PAH patient who had undergone combined heart and lung transplantation.
  • Bcl-xL expression Having observed an increased level of Bcl-xL expression in PAH patient samples and in PAH rat lungs, the inventors were keen to investigate alternative splicing of the Bcl-x gene in cells including PASMCs and ECs, which are involved in the remodelling of small pulmonary arteries.
  • the inventors first investigated the expression of Bcl-x transcripts in human PASMCs. These cells were isolated from explanted lung samples from a PAH patient harbouring a pathogenic mutation (p.R899X) in the BMPR1 gene. PAH-PASMCs and commercially available wild-type PASMCs were grown in tissue culture plates until confluency and their total RNAs were isolated.
  • BMPRII mediated signalling elicits pro-apoptotic effect in PASMCs.
  • K-l mice harbouring the PAH-associated BMPR2 nonsense mutation (p.R899X). These mice were asymptomatic at the age of three months but developed age-related PAH at the age of six months (4) .
  • PASMCs were isolated from asymptomatic wild-type ( bmpr2 +/+ ) and mutant (, bmpr2 R899X+/ -) mice and the rate of apoptosis was determined by measuring cysteinyl aspartate proteases (caspase) activity using commercially available kits (Promega) following manufacturer’s instructions. Consistent with previous observations, the basal activity level of caspases 3 and 7 was attenuated in mutant cells compared with wild-type ( Figure 3D), the effect of BMPR2 mutation on apoptosis in mouse PASMCs as determined by caspase 3/7 activities. PASMCs were derived from wild-type and mutant mice harbouring the pathogenic BMPR2 mutation (p.R899X).
  • the inventors further characterized the pro-apoptotic effect of BMP signalling on ST- induced apoptosis by stimulating the cells with BMP9 ligand (Figure 3E).
  • the caspase activity was further increased following ligand stimulation, which is consistent with the previous finding of BMP-dependent caspase activation.
  • the pro-apoptotic effect of BMP stimulation was absent in BMPRII deficient cells. In these cells, BMP9 inhibited staurosporine induced cell death in a dose dependent manner (Figure 3E).
  • BMPRII dysfunction potentiates the apoptotic-resistant phenotype through the upregulation of Bcl-xL, and that selective inhibition of Bcl-xL induces apoptosis (Figure 3F).
  • the inventors used PAH-PASMCs harbouring the BMPR2 (p.R899X) mutation as these cells showed reduced level of apoptosis compared with wild-type.
  • PASMCs were treated with 2, 3 DCPE hydrochloride for 48 hours and the caspase activity was determined.
  • the compound significantly increased apoptosis in PAH-PASMCs in a concentration dependent manner (Figure 3G).
  • it also induced apoptosis in wild- type PASMCs these results show the selective inhibition of Bcl-xL by a chemical agent induces apoptosis in PAH-PASMCs.
  • the inventors selected the human embryonic kidney (HEK293T) cells as the transfection efficiency is higher in these cells compared with PASMCs and PAECs.
  • Cells were co-transfected with a plasmid harbouring the wild-type BMPRII receptor.
  • the inventors found that the expression of Bcl-xL transcripts was increased in cells overexpressed with the BMPRII receptor compared with the untreated control ( Figure 4E-F).
  • HEK293T cells treated with BMP9 ligand (10ng/ml) increased the level of Bcl-xL transcripts, the ligand elicited no discernible effect on the preferential expression of Bcl-x in PAECs (Figure 4G-H).
  • the inventors were interested to investigate the effect of a wide range of mutations identified in PAH patients on pro-survival activity. To investigate this, the inventors employed HEK293T cells as in these cells BMPRII-mediated signalling increased the expression of pro-survival Bcl-xL isoform.
  • the pro-survival effect of BMPRII-mediated signalling was investigated by transfecting HEK293T cells with plasmids harbouring either BMPRII wild-type or a wide range of mutant receptors (Figure 4J). Cell viability was measured using Cell-Titre Glo Cell Viability Assay Kit (Promega) following manufacturer’s instructions.
  • BMP signalling promoting activity of BMP4 ligand and BMPRII receptor was tested using the BMP-responsive 3GC2-Lux reporter assay as previously described.
  • HEK293T cells were stimulated with the ligand (10ng/ml) at various time points (30 mins, 1 h, 3hrs, 5hrs, 7hrs, 9hrs and 24hrs) with and without BMPRII overexpression and their luciferase and b-galactosidase activities were determined. Reporter activation was observed after 7 hours of ligand stimulation and the highest activity was observed at the 24 hour time point ( Figures 5C-E). The BMP signalling promoting activity of BMP4 ligand and BMPRII receptor.
  • FIG. 5C Diagram depicting the BMP signalling cascade as determined by the BMP responsive 3GC2-Lux reporter assay.
  • the luc-gal activities of cells at Oh time point were set as 100. **P ⁇ 0.01 , ***P ⁇ 0.001 compared with control treated for 0 hour. Together, these results indicate that the attenuation of caspase activities observed in these cells might be due to the activation of BMP signalling.
  • the rate of apoptosis was determined using the Caspase-Glo ® 3/7 Assay (Promega). ***P ⁇ 0.001 compared with untreated control, NS-non significant. Results show that BMP signalling inhibits caspase 3/7 activities via the ALK1 receptor
  • BMPRII and ALK1 receptors work synergistically to activate the BMP signalling pathway
  • the inventors then investigated the effects of ligands including BMP4 and BMP9 on BMPRII receptor (Figure 6B).
  • the inventors found that HEK293T cells co expressing BMPRII and stimulated with either BMP4 or BMP9 showed increased reporter activation compared with cells expressing BMPRII alone ( Figure 6B).
  • BMP9 but not BMP4 ligand significantly increased ALK1-BMPRII-mediated reporter activation compared with cells transfected with BMPRII and stimulated with either BMP4 or BMP9 ligand ( Figures 6C-D).
  • ALK1-BMPRII-mediated reporter activation was significantly increased compared with cells stimulated with either of BMP4 or BMP9 ligands.
  • the luc-gal activity of ligand stimulated cells was set as 100. ***P ⁇ 0.001 compared with ligand stimulated control, NS-non significant.
  • the p.D485G mutant was unable to interact with either ALK3 or ALK6 receptor and failed to activate the BMP-responsive reporter in the absence and presence of ALK3 and ALK6 receptor overexpression and BMP4 ligand stimulation.
  • FIG. 6C shows mutation in the kinase domain of BMPRII receptor (p.D485G) significantly reduced BMP9-ALK1- mediated reporter activation.
  • the luc-gal activity of BMP9 ligand stimulated cells was set as 100. ***P ⁇ 0.001 compared with ligand stimulated control. Results show mutations in either ALK1 or BMPRII receptor impair the BMPRII-ALK1 pathway
  • the activation domain was fused with the intracellular part of the BMPRII receptor, whilst the ALK1 lacking the extracellular domain was fused with DNA binding domain (DBD).
  • the dual-reporter was co-transfected with both the ALK1-DBD and BMPRII-AD.
  • the cells generate only the upstream b-galactosidase protein ( Figure 7A).
  • Figure 7A In the event of BMPRII-ALK1 interactions, both luciferase and b-galactosidase activities are generated.

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Abstract

La présente invention concerne de nouveaux biomarqueurs destinés au diagnostic et au pronostic de l'hypertension artérielle pulmonaire. Plus particulièrement, l'invention concerne l'utilisation de Bcl-xL et/ou de Bcl-xS ou de rapports entre ceux-ci en tant que biomarqueurs pour l'hypertension artérielle pulmonaire, l'invention comprend des procédés de diagnostic et de pronostic de l'hypertension artérielle pulmonaire à l'aide des biomarqueurs et des interventions thérapeutiques pour l'hypertension artérielle pulmonaire.
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