Patent Application
20190031773
This application claims priority to EP Patent Application No. 18165811.3 filed 5 Apr. 2018, and EP Patent Application No. 17172756.3 filed 24 May 2017, the entire contents of each of which are hereby incorporated by reference.
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 9, 2018, is named 6487-0206_SL.txt and is 27,663 bytes in size.
The present invention provides inhibitors and antagonists of human PYCR1, for the treatment and/or prevention of a neoplastic disease. The invention provides inhibitors and antagonists in the form of antibodies, nucleic acids, aptamers, or small molecules. The invention also provides assays and screening technologies to find such inhibitors and antagonists.
Despite tremendous progress in the last 3 decades, cancer therapy is still a field with many unmet needs. There are cancer types which do not respond on established therapies, or respond poorly, with high recurrence rates or continued formation of metastases. This applies, inter alia, to tumors and cancers in the liver, the skin or the oesophagus.
With an estimated 700,000 new cases per year, hepatocellular carcinoma (HCC) represents the fifth commonest cancer and is the third leading cause of cancer death worldwide. HCC cases currently occur predominantly in East and Southeast Asia, and Sub-Saharan Africa due to Hepatitis C viral infection and are increasing in the Western nations, largely due to an increasing incidence of metabolic complications, such as non-alcoholic steatohepatitis related to obesity and type II diabetes. The 5-year survival rate remains below 12% due to limited treatment options (El-Serag 2011). Besides Sorafenib, an oral multikinase inhibitor approved for the treatment of HCC (Llovet, Ricci et al. 2008), there is no other pharmacological agent approved for therapy of HCC. However, due to the severe side effects and only a 2-3 month gain of life, the identification of additional effective therapeutic targets remains a top priority.
To meet increased energetic and anabolic needs of sustained cell growth, cancer cells undergo dramatic metabolic reprogramming (Boroughs and DeBerardinis 2015). The expression changes observed in cancer cells can be linked to the activity of multiple classic oncogenes and tumor suppressors. Overall, accumulating evidence demonstrates that changes in metabolism can confer a potent influence on tumor development and growth, enough so that it is now considered an emerging hallmark of cancer (Ward and Thompson 2012). These metabolic changes also hold the potential for therapeutic intervention. A notable advancement has been the targeting of changes in serine and glycine metabolism for certain cancers (Locasale, Grassian et al. 2011; Possemato, Marks et al. 2011).
Therefore, metabolic enzyme expression profiles in different HCC animal models were compared to identify metabolic pathways that could be targeted offering treatment options for HCC patients. In vitro gene silencing experiments revealed similar results for HCC, melanoma and oesophageal cancer cell lines.
These and further objects are met with methods and means according to the independent claims of the present invention. The dependent claims are related to specific embodiments.
Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts or structural features of the devices or compositions described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include singular and/or plural referents unless the context clearly dictates otherwise. Further, in the claims, the word “comprising” does not exclude other elements or steps. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.
It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.
It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the length of this specification manageable. It is further to be understood that the content of the prior art documents referred to herein is incorporated by reference, e.g., for enablement purposes, namely when e.g. a method is discussed details of which are described in said prior art document. This approach serves to keep the length of this specification manageable.
According to one aspect of the invention, an antagonist of PYCR1 for the treatment and/or prevention of a neoplastic disease is provided.
The method disclosed herein to determine such antagonist is so comprehensive and sufficiently disclosed that it allows the identification of such antagonist without the need to embark on an unduly burdensome research program, in particular when the skilled person recourses to common general knowledge. This is because the specification provides sufficient information on how to select such potentially active molecule both for its theoretical use to treat neoplastic disease, as well as for its therapeutic suitability.
Pyrroline-5-carboxylate reductase 1 (PYCR1) is a mitochondria-associated enzyme which belongs to the family of oxidoreductases. Preferably, said PYCR1 is human PYCR1. In humans, the enzyme is encoded by the PYCR1 gene (PYCR1, UniProt: P32322). The enzyme catalyzes the NAD(P)H-dependent conversion of pyrroline-5-carboxylate to proline. The reverse reaction is catalyzed by proline dehydrogenase (PRODH).
The enzyme forms a homopolymer and localizes to the mitochondrion. Alternate splicing of the gene results in different transcript variants encoding different isoforms, as disclosed in Entrez Gene: PYCR1 pyrroline-5-carboxylate reductase 1. Homozygous deletion or missense mutations of PYCR1 have been shown to cause skin disorders in humans like Autosomal recessive cutis laxa (ARCL) (Reversade et al (2009), Kretz et al (2011). It has further been shown that expression of PYCR1 is upregulated in a variety of tumor types (Roth et al).
However, upregulation does not mean that PYCR1 is causative for the genesis of cancer in general, or specific tumors in particular. Upregulation can also be the downstream consequence of upregulation of another gene, or caused by a general impairment of gene regulation as it frequently occurs in malignant tissues.
The inventors have for the first time shown that inhibition of PYCR1 can reduce proliferation in specific tumor models in vitro and in vivo, particularly in liver tumor, skin tumor, and esophageal tumor.
While these findings have been made with gene knockdown experiments, part of which were induced knockdowns in tumor xenograft mice, an extrapolation of these findings on potential effects of an antagonist or inhibitors of PYCR1, e.g., a suitable antibody, a suitable nucleic acid molecule, a suitable aptamer or a suitable small molecule, is plausible. The findings are quite surprising insofar as homozygous deletion or missense mutations of PYCR1 have been shown to cause skin disorders in humans like Autosomal recessive cutis laxa (ARCL). Hence, inhibition of PYCR1 has so far been associated with pathologic effects rather than with a therapeutic approach for treating cancer.
Accordingly, in one embodiment of the invention, a liver tumor, or a secondary tumor derived from a liver tumor. This includes hepatocarcinoma, hepatocellular carcinoma, cholangiocarcinoma, angiosarcoma and/or hepatoblastoma and other liver tumors.
In one other embodiment of the invention, the neoplastic disease treated by the PYCR1 antagonist is a skin tumor, or a secondary tumor derived from a skin tumor. This includes melanoma, basal-cell skin cancer, squamous-cell skin cancer or merkel cell carcinoma, and other skin tumors.
In one other embodiment of the invention, the neoplastic disease treated by the PYCR1 antagonist is an oesophageal tumor, or a secondary tumor derived from an oesophageal tumor.
As used herein the term “antagonist of PYCR1 “refers to regulators or effectors or modulators of PYCR1 nucleic acids that inhibit, decrease or prevent the intracellular response of PYCR1 polypeptides or PYCR1 nucleic acids (including mRNA and genomic DNA). The term is used interchangeably with the term “inhibitor”.
According to one embodiment, the antagonist inhibits, directly or indirectly, PYCR1-mediated formation of L-Proline. In this context, the term “directly” means that the inhibitor according to the invention directly affects the enzymatic activity of PYCR1, e.g., by binding to the latter and/or steric hindrance. The term “indirectly” means that the inhibitor affects the gene expression of PYCR1, e.g., by sequence-specific interaction with the mRNA or the encoding genomic DNA.
According to another embodiment of the invention, the antagonist is a monoclonal antibody, or a target-binding fragment or derivative thereof retaining target binding capacities that specifically binds to one or more isoforms of the PYCR1 protein.
As used herein, the term “monoclonal antibody (mAb)”, shall refer to an antibody composition having a homogenous antibody population, i.e., a homogeneous population consisting of a whole immunoglobulin, or a fragment or derivative thereof retaining target binding capacities. Particularly preferred, such antibody is selected from the group consisting of IgG, IgD, IgE, IgA and/or IgM, or a fragment or derivative thereof retaining target binding capacities.
As used herein, the term “fragment” shall refer to fragments of such antibody retaining target binding capacities, e.g.
As used herein, the term “derivative” shall refer to protein constructs being structurally different from, but still having some structural relationship to, the common antibody concept, e.g., scFv, Fab and/or F(ab)2, as well as bi-, tri- or higher specific antibody constructs, and further retaining target binding capacities. All these items are explained below.
Other antibody derivatives known to the skilled person are Diabodies, Camelid Antibodies, Nanobodies, Domain Antibodies, bivalent homodimers with two chains consisting of scFvs, IgAs (two IgG structures joined by a J chain and a secretory component), shark antibodies, antibodies consisting of new world primate framework plus non-new world primate CDR, dimerised constructs comprising CH3+VL+VH, and antibody conjugates (e.g. antibody or fragments or derivatives linked to a toxin, a cytokine, a radioisotope or a label). These types are well described in literature and can be used by the skilled person on the basis of the present disclosure, without adding further inventive activity.
As discussed above, PYCR1 is sufficiently specified to enable a skilled person to make a monoclonal antibody thereagainst. Routine methods encompass hybridoma, chimerization/humanization, phage display/transgenic mammals, and other antibody engineering technologies.
Methods for the production of a hybridoma cell are disclosed in Köhler & Milstein (1975). Essentially, e.g., a mouse is immunized with a human PYCR1 protein, following B-cell isolation and fusion with a myeloma cell.
Methods for the production and/or selection of chimeric or humanised mAbs are known in the art. Essentially, e.g., the protein sequences from the murine anti PYCR1 antibody which are not involved in target binding are replaced by corresponding human sequences. For example, U.S. Pat. No. 6,331,415 by Genentech describes the production of chimeric antibodies, while U.S. Pat. No. 6,548,640 by Medical Research Council describes CDR grafting techniques and U.S. Pat. No. 5,859,205 by Celltech describes the production of humanised antibodies.
Methods for the production and/or selection of fully human mAbs are known in the art. These can involve the use of a transgenic animal which is immunized with human PYCR1, or the use of a suitable display technique, like yeast display, phage display, B-cell display or ribosome display, where antibodies from a library are screened against human PYCR1 in a stationary phase.
In vitro antibody libraries are, among others, disclosed in U.S. Pat. No. 6300064 by MorphoSys and U.S. Pat. No. 6,248,516 by MRC/Scripps/Stratagene. Phage Display techniques are for example disclosed in U.S. Pat. No. 5,223,409 by Dyax. Transgenic mammal platforms are for example described in EP1480515A2 by TaconicArtemis.
IgG, scFv, Fab and/or F(ab)2 are antibody formats well known to the skilled person. Related enabling techniques are available from the respective textbooks.
As used herein, the term “Fab” relates to an IgG fragment comprising the antigen binding region, said fragment being composed of one constant and one variable domain from each heavy and light chain of the antibody
As used herein, the term “F(ab)2” relates to an IgG fragment consisting of two Fab fragments connected to one another by disulfide bonds.
As used herein, the term “scFv” relates to a single-chain variable fragment being a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker, usually serine (S) or glycine (G). This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide.
Modified antibody formats are for example bi- or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates and the like. These types are well described in literature and can be used by the skilled person on the basis of the present disclosure, with adding further inventive activity.
Finding a suitable antibody, or fragment or derivative, that is capable of acting as an antagonist of PYCR1, e.g., by binding to its active centre, is hence a matter of routine for the skilled person, based on the public availability of the amino acid sequences of the different PYCR1 isoforms.
Polyclonal antibodies against PYCR1 for scientific research are commercially available, e.g., from Proteintech (20962-1-AP and 13108-1-AP), Abcam (ab72953), ThermoFischer (PA5-30141, PA5-26890), Sigma Aldrich (HPA047660, sab2104620), Novus Biologicals (H00005831-B01P and NBP1-57934)), hence demonstrating that the skilled person is capable of making a therapeutic antibody against PYCR1.
Because PYCR1 is an intracellular enzyme, the antibody or its fragment or derivative needs to be funneled or trafficked into the intracellular space. Routine technologies are available for these purposes, which are disclosed, inter alfa, in Chen & Erlanger (2002), Berguig et al (2015), Meunier, Executive Interview, Thura et al (2016).
According to another embodiment of the invention, the antagonist comprises a first nucleic acid molecule that specifically binds to a second nucleic acid molecule, which second nucleic acid molecule encodes an isoform of the PYCR1 protein.
Said second nucleic acid molecule can be an mRNA transcribed from the gene encoding for the PYCR1 protein. Said second nucleic acid is devoid of introns, but due to alternative splicing different mRNAs transcribed from the gene encoding for the PYCR1 protein can differ from one another. In such case, the first nucleic acid molecule can be a siRNA (small interfering RNA) or a shRNA (short hairpin RNA).
siRNAs are short artificial RNA molecules which can be chemically modified to enhance stability. Because siRNAs are double-stranded, the principle of the ‘sense’ and the ‘antisense’ strand also applies. The sense strands have a base sequence identical to that of the transcribed mRNA and the antisense strand has the complementary sequence. Technically, a siRNA molecule administered to a patient is bound by an intracellular enzyme called Argonaut to form a so-called RNA-induced silencing complex (RISC). The antisense strand of the siRNA guides RISC to the target mRNA, where the antisense strand hybridizes with the target mRNA, which is then cleaved by RISC. In such way, translation of the respective mRNA is interrupted. The RISC can then cleave further mRNAs. Delivery technologies are e.g. disclosed in Xu and Wang (2015). Finding a suitable sequence for the siRNA is a matter of routine for the skilled person, based on the public availability of the different mRNA isoforms of PYCR1.
shRNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). shRNA can be delivered to cells, e.g., by means of a plasmid or through viral or bacterial vectors. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover. The respective plasmids comprise a suitable promoter to express the shRNA, like a polymerase III promoter such as U6 and H1 or a polymerase II promoter. Once the plasmid or vector has integrated into the host genome, the shRNA is transcribed in the nucleus. The product mimics pri-microRNA (pri-miRNA) and is processed by Drosha. The resulting pre-shRNA is exported from the nucleus by Exportin 5. This product is then processed by Dicer and loaded into the RNA-induced silencing complex (RISC), after which the same silencing follows as in siRNA. Finding a suitable sequence for the shRNA is a matter of routine for the skilled person, based on the public availability of the different mRNA isoforms of PYCR1.
Said second nucleic acid molecule can also be a genomic DNA comprised in the gene encoding for the PYCR1 protein. Said gene comprises several non-coding introns, hence its sequence differs from the sequence of the mRNA or the cDNA disclosed herein.
In such case, the first nucleic acid molecule can be the guide RNA of a CRISPR Cas system (see, e.g., Jinek et al (2012)), which guide RNA comprises a target-specific crRNA (“small interfering CRISPR RNA”) capable of hybridizing with a genomic strand of the PYCR1 gene (or, the first nucleic acid molecule can be the crRNA alone). The guide RNA/crRNA is capable of directing the Cas enzyme, which is an endonuclease, to the PYCR1 gene, where the Cas enzyme carries out sequence specific strand breaks. By creating one or more double strand breaks, the PYCR1 gene hence can be silenced. To use said system for in vivo gene silencing of PYCR1, e.g., in different cells of a tumor, a dedicated delivery technology is required, which comprise a delivery vehicle such as lipid nanoparticles, as for example discussed in Yin et al (2016). Finding a suitable sequence for the crRNA comprised in the guide RNA is a matter of routine for the skilled person, based on the public availability of the genomic sequence of the PYCR1 gene.
In another embodiment, said first nucleic acid molecule can also the guide RNA of a CRISPR Cpf system (Zetsche et al (2015)), which guide RNA comprises a target-specific crRNA (“small interfering CRISPR RNA”) Similar to CRISPR Cas, the guide RNA is capable of directing the Cpf enzyme, which is an endonuclease, to the PYCR1 gene. As regards technical considerations, e.g., delivery for in vivo applications and finding of the suitable sequence for the first nucleic acid molecule, similar aspects as with CRISPR Cas apply.
Further embodiments of the CRISPR technology are currently under development, with different endonucleases. However, all these approaches use a target-specific RNA (the guide RNA or crRNA as in CRISPR Cas) that hybridizes with a target sequence. In all these cases, the target-specific RNA qualifies as the first nucleic acid molecule in the meaning of the preferred embodiment discussed herein. As regards technical considerations, e.g., delivery for in vivo applications and finding of the suitable sequence for the first nucleic acid molecule, similar aspects as with CRISPR Cas apply.
According to another embodiment of the invention, the antagonist is an aptamer that specifically binds to one or more isoforms of the PYCR1 protein, and/or inhibits the activity thereof.
Aptamers are oligonucleotides that have specific binding properties for a pre-determined target. They are obtained from a randomly synthesized library containing up to 1015 different sequences through a combinatorial process named SELEX (“Systematic Evolution of Ligands by EXponential enrichment”). Aptamer properties are dictated by their 3D shape, resulting from intramolecular folding, driven by their primary sequence. An aptamer3D structure is exquisitely adapted to the recognition of its cognate target through hydrogen bonding, electrostatic and stacking interactions. Aptamers generally display high affinity (Ka about micromolar for small molecules and picomolar for proteins).
An overview on the technical repertoire to generate target specific aptamers is given, e.g., in Blind and Blank (2015). Aptamers can also be delivered into the intracellular space, as disclosed in Thiel & Giangrande (2010).
Finding a suitable aptamer that is capable of acting as an antagonist of PYCR1, e.g., by binding to its active centre, is hence a matter of routine for the skilled person, based on the public availability of the amino acid sequences of the different PYCR1 isoforms.
According to another embodiment of the invention, the antagonist is a polypeptide or protein fragment, such as a synthesized polypeptide and a stapled peptide, that specifically binds to one or more isoforms of the PYCR1 protein, and/or inhibits the activity thereof.
An overview on the technical repertoire to generate stapled peptides is given, e.g., in Blind and Blank (2015) and Walensky and Bird (2014).
Finding a suitable polypeptide or protein fragment that is capable of acting as an antagonist of PYCR1, e.g., by binding to its active centre, is hence a matter of routine for the skilled person, based on the public availability of the amino acid sequences of the different PYCR1 isoforms.
According to another embodiment of the invention, the antagonist is a small molecule that specifically binds to one or more isoforms of the PYCR1 protein, and/or inhibits the activity thereof.
Said antagonist can for example be found by means of a PYCR1 inhibition assay, where the impact of a candidate molecule on the PYCR1-catalyzed transformation from L-Proline to L-1 pyrroline-5-carboxylate or L-1 pyrroline-5-carboxylate to L-Proline is determined. An example for such assay may look as follows:
The assay is based on the ability of PYCR1 to catalyze the oxidation of L-proline to L-1 pyrroline-5-carboxylate which occurs concomitant with the reduction of the cosubstrate NAD+ to its reduced form NADH+H+. The amount of NADH+H+ was quantified using the NAD(P)H-Glo™ kit from Promega (catalogue number G9062). Components of the kit generate a luciferase-based luminescence signal the intensity of which is linearly proportional to the amount of NADH+H+ generated by the PYCR1 reaction and consequently the PYCR1 activity. The luminescence intensity was taken as the measure for the amount of NADH+H+ generated by PYCR1. Inhibition of PYCR1 results in a decreased amount of NADH+H+ as compared to an uninhibited control reaction and a consequently decreased luminescence intensity Luminescence intensity was measured using a ViewLux Imager (PerkinElmer). The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition) and IC50 values were calculated using Genedata Screener® software.
Based on the above description, in particular when using the said assays, the skilled person is able to identify suitable PYCR1 antagonists or inhibitors. Examples of small molecular PYCR1 antagonists or inhibitors identified with said PYCR1 Inhibition Assay are shown in the following table, with the IC50 indicated as determined in the PYCR1 Inhibition Assay.
Generally, an antagonist or inhibitor identified by an assay described herein can be further validated for therapeutic effect by administration to a tumor or tumor model in which the PYCR1 gene is overexpressed and/or there is excess of PYCR1 protein. A decrease in tumor growth confirms therapeutic utility of the modulator. Prior to treating a patient with the antagonist or inhibitor, the likelihood that the patient will respond to treatment can be diagnosed by obtaining a tumor sample from the patient, and assaying for expression of PYCR1. The expression data for the gene can also be used as a diagnostic marker for disease progression. The assay can be performed by expression analysis, by an antibody directed to the gene target or its expression product, or by any other available detection method.
According to another embodiment of the invention, the PYCR1 protein to which the antibody, aptamer or small molecule binds comprises a consensus sequence according to SEQ ID No 10. Said consensus sequence refers to SEQ ID Nos 6-8, which are the different isoforms of the PYCR1 enzymes.
According to another embodiment of the invention, the PYCR1 protein to which the antibody, aptamer or small molecule binds comprises a sequence according to any of SEQ ID Nos 6-8.
According to another embodiment of the invention, the nucleic acid encoding an isoform of the PYCR1 protein comprises a consensus sequence according to SEQ ID No 9. Said consensus sequence refers to SEQ ID Nos 1-5, which are the different transcription variants of the PYCR1 encoding gene.
According to another embodiment of the invention, the nucleic acid encoding an isoform of the PYCR1 protein comprises a sequence according to any of SEQ ID Nos 1-5.
According to another embodiment of the invention, the neoplastic disease is characterized by overexpression of PYCR1 and/or reduced expression of PRODH. The term PRODH refers to proline dehydrogenase, which catalyzes the reverse reaction of PYCR1. Preferably, said PRODH is human PRODH (UniProtKB-O43272).
As used herein, the terms “overexpression of a gene” and “reduced expression of a gene” refer to expression patterns and/or expression levels of a gene normally not occurring under natural conditions, e.g. in a healthy tissue. The respective comparative expression patterns or levels can e.g. be taken from predetermined average expression patterns or levels from tissue databases or the like.
According to another embodiment of the invention, the neoplastic disease is characterized by excessive levels of PYCR1 protein (in the tumor/tumor cells).
As used herein, the terms “excess of a protein” and “excessive level of a protein” or the like refer to levels or amounts of a protein normally not occurring under natural conditions, e.g. in a healthy tissue. The respective comparative levels or amounts can e.g. be taken from predetermined average levels, levels/amounts from tissue databases, levels/amounts from non-tumor tissue (e.g. healthy tissue) or the like.
According to another aspect of the invention, the use of the antagonist (for the manufacture of a medicament) in the treatment of a human or animal subject being diagnosed for, suffering from or being at risk of developing a neoplastic disease, or in the prevention of such condition, is provided.
The invention further provides a pharmaceutical composition comprising an antagonist according to the above description, a combination of such pharmaceutical composition and one or more further therapeutically active compounds, and a method for treating or preventing a disorder or condition associated with the undesired expression of PYCR1, comprising administering to a subject in need thereof an effective amount of the said antagonist, pharmaceutical composition or combination.
While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. Any reference signs should not be construed as limiting the scope. All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5′->3′.
The experiments shown herein clearly support PYCR1 as a target whose inhibition provides a therapy option for HCC. PYCR1 is overexpressed in rat and mouse HCC models compared to normal and regenerating liver as well as in human HCC cell lines compared to human hepatocytes, suggesting a role in tumorigenesis. Suppression of PYCR1 in tumor cell lines that show increased PYCR1 was found to cause a dramatic decrease in tumor cell proliferation. Short hairpin RNAs (shRNAs) that inhibit PYCR1 expression caused a change in morphology of MH3924A cells and extensively reduced tumor growth of subcutaneous xenografts generated with shRNA transfected MH3924A cells. Moreover, doxycycline-inducible PYCR1 shRNAs caused reduction of tumor growth in orthotopic MH3924A xenograft models in either the preventive setting where PYCR1 knockdown is induced one day after cell inoculation or in the therapeutic setting where PYCR1 knockdown is induced after tumor formation, i.e. 7 days after cell inoculation to degrees consistent with their capacity to suppress PYCR1.
Based on the knockdown experiments shown herein, the suitability of a
All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the Biomedical Research Council of Singapore, carried out according to the guidelines for the care and use laboratory animals and BRC IACUC guidelines for cancer research in mice and rats.
Mouse and rat amino acid metabolism I/II PCR arrays of 96-well plate format (cat. No.PAMM-130Z, cat. No.PARN-130Z) were purchased from Qiagen. Total RNAs from diethylnitrosamine-induced mouse liver tumors, orthotopic rat morris hepatoma tumors, regenerating liver tissue, and non-tumor liver tissues were extracted using RNeasy mini kit (Qiagen). 1.2 μg of total RNA was used for first strand synthesis with RT2 First Strand Kit (Qiagen), then first strand reaction mixed with RT2 SYBR green Mastermix was evenly distributed into 96 wells plate format containing the predispensed gene specific primer sets. Quantitative real-time PCR was performed according to RT2 Profiler PCR Array Handbook in an ABI StepOnePlus instrument and data were normalized to the 5 housekeeping genes (ACTB, B2M, GAPDH, HSP90ab1, GUSB) using the ΔΔCT method. The PCR conditions were 95° C. for 10 min for initial enzyme activation and 40 cycles of amplification at 95° C. for 15 s and 60° C. for 1 min. Ct values were exported for web-based data analysis at http://perdataanalysis.sabiosciences.com/per/arrayanalysis.php. In DEN-induced mouse tumors and orthotopic rat MH3924A tumors the genes for the proline biosynthesis enzymes ALDH18A1 and PYCR1 were upregulated and those for the proline degradation enzymes PRODH and OAT were downregulated compared to normal liver tissue and regeneration liver tissue (
Array Northern is a proprietary application providing an overview of the expression of genes across healthy and diseased tissues. Array Northern is based on a compilation of Affymetrix gene expression experiments (HG-U133_Plus_2 array) from in-house studies and publicly available studies which were downloaded from Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) and ArrayExpress (https://www.ebi.ac.uk/arrayexpress/). Affymetrix CEL files were processed with the Affymetrix MAS5 algorithm and median-normalized. Samples were annotated with a controlled vocabulary for tissues, cell types and diseases. Array Northern generates box plots where each box summarizes the expression data of all samples of a predefined combination of sample annotation categories. The expression of the PYCR1 gene probe 202148_s_at is displayed for the data collection “All Diseased, Tumor, Normal samples for AN 2012-09” and for the data collection “Human Cell Lines for AN 2012-08-14”. Data for tumor indications with increased PYCR1 expression are shown in
Short hairpin oligonucleotides targeting rat Pycr1 were subcloned into lentiviral vector pLKO or pLKO-Tet via Agel and EcoRl sites to generate respective lentiviral knockdown constructs. All the DNA constructs were validated by sequencing. The following oligonucleotides were used for vector construction: Rat PYCR1.1: GCT GCC ATA AAG AAG ACT GTA; Rat PYCR1.2: GAA CCT GAC ACC CCA CAA CAA; Rat PYCR1.3: GGC AGC CAA GAT GCT ACT AGA; Lentiviral particles were produced by co-transfection of HEK293T cells with lentiviral constructs and third generation packaging plasmids using the calcium phosphate transfection method. The supernatant containing the viral particles was collected 72 h after transfection, centrifuged at 1,000 g for 5 min and filtered with 0.45 μm filter. Collected lentiviruses were stored at −80° C.
Morris hepatoma cells (MH3924A) were cultured in Dulbecco's modified eagle medium (DMEM) media supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 μg/ml streptomycin. To assess cell proliferation, cells were seeded in T25 flasks and incubated in DMEM complete medium for 24 hours to allow attachment. Subsequently, seeding cells were infected with shRNA lentivirus in the presence of 8μg/ ml of polybrene for about 7 hours, cells were grown for varying lengths of time, at the end of which they were detached by trypsinization and counted with Countess (Life Technologies, Carlsbad, USA).
Total RNA was extracted from tumor and nontumor liver tissues according to the manufacturer's protocol (RNeasy kit, Qiagen, Germany). Total RNA (1.2 μg) was then reverse transcribed to cDNA by Superscript III reverse transcriptase (Invitrogen) with random hexamer in a total reaction volume of 20 μl. Real-time PCR reactions (in triplicates) were performed with the Fast Real-Time PCR System (Applied Biosystems, USA), using 1 μl of cDNA product in a reaction volume of 10 μl. The Taqman primers and probes (Applied Biosystemis) for target genes, Pycr 1 (Rn01751579_m1, Mm01205845_m1), Prodh (Rn01404902_m1, Mm00448401_m1), the endogenous control genes were rat Gapdh (4352338E, VIC lavelled), mouse Hprt: (Mm 00446968 VIC labelled). The PCR conditions were 95° C. for 20 s and 40 cycles of amplification at 95° C. for 3 s and 60° C. for 30 s. The transcript levels were expressed as fold of changes compared with non-tumor samples unless stated otherwise (
For tumor and non-tumor tissue proteins (
Female scid mice (InVivos, Singapore) were subcutaneously inoculated with 1×106rat MH3924A cells that were stably transfected with either shRNA Pycr1.2 or scrambled control shRNA. Mice were sacrificed and tumors excised 3 weeks later. Tumor weight was significantly reduced in the group that was inoculated with MH3924A cells stably expressing shRNA Pycr1.2 compared to control (n=5).
The HCC model was created by the direct intrahepatic injection of rat MH3924A cells harboring inducible shRNA Pycr1.2 or scrambled shRNA constructs into scid mice (InVivos, Singapore). The mice were anesthetized and a midline incision was made to expose the liver. The cells were then slowly injected under the hepatic capsule into the upper left lobe of the liver using a 27-G needle. A pale, whitish color could be observed at the point of injection under the hepatic capsule. Gentle compression was applied with a cotton applicator to stop bleeding and reflux of the cells. The abdomen was closed with a 4-0 silk suture.
In the preventive setting (
In the therapeutic setting, mice were subjected to liver MRI (magnetic resonance imaging) 7 days (1st experiment,
2-week-old male mice were injected intraperitoneally with 50 mg/kg body weight diethylnitrosamine (DEN). Mice were sacrificed at end of 8 months to collect liver tumor and perform gene expression analysis (
Hepatectomy surgery was based on the method published by (Mitchell and Willenbring 2008). Wild type C57BL/6 mice or ACI rats were deeply anesthetized with Ketamine/xylazine via i.p. injection, laparotomy was performed to exposed liver, 4-0 silk threads were placed on the base of left lobe and median lobe, sutures were tied, then curved scissors were used to cut the tied left and median lobe. Liver regeneration was monitored on different time points, on 16th day, the liver weight almost recovered to its original size. To identify controlled cell proliferation, we collected liver tissues 48 hr after hepatectomy to analyse gene expression, this was to avoid the acute response soon after hepatectomy.
shRNA's:
xCelligence:
All cell lines used in the following experiments are available from ATCC (American Type Culture Collection, Manassas, Va. 20110-2209). shRNA expressing lentiviruses were produced at the NMI, Kusterdingen, Germany 1 million cells in 1 ml medium/well were seeded in a 6well plate and 25 μl of lentivirus expressing non-targeted or PYCR1 specific shRNAs (listed below) were added 6 hours later. The following day (day 1), the cells were washed with PBS, trypsinated and 1.5-5 million cells were seeded in 2 wells of a 6well culture plate for lysate generation and 2000 cells in 150 μl medium per well were seeded in triplicates in a 96we11 xCELLigence E-plate to monitor proliferation for about 10 days (OMNI Life Science). Non-treated cells were used as control. On day 3 or 4, the medium was exchanged and 0.6μg/ml puromycin was added to select for shRNA expressing cells.
On day 4 and day 8, 9 or 11, lysates were generated from the control and transfected cells grown in 6 well plates using 50 μl of RIPA buffer/well, followed by protein determination (Protein Assay Reagent) and 40 μg lysate protein/lane were separated by SDS-PAGE and blotted using the iBlot system.
For PYCR1 knockdown confirmation, the blot membrane was blocked with 2% nonfat milk powder, washed with TBST buffer and incubated with the PYCR1 specific antibody over night at 4° C. After washing 3 times with TBST for 5 min each the blot membrane was incubated for 45 min with the secondary antibody and after washing again 3 times with TBST for 5 min and once with PBS, the blot was analyzed using the Odyssey Imaging System (LI-COR Biotechnology-GmbH).
The human liver carcinoma cell lines SNU-398, HepG2 and Hep3B2 as well as the rat Morris Hepatoma cell line MH3924A and the non-tumorigenic HaCaT cell line were transfected with 2 PYCR1 shRNAs and a non-targeted shRNA as control and proliferation was monitored in comparison to untreated cells using the xCELLigence system (OMNI Life Science). Knockdown was confirmed by Western blot on days 4 and 8 or 11 after transfection. PYCR1 knockdown results in anti-proliferative effects in the liver cancer cell lines while PYCR1 silencing did not significantly inhibit proliferation or cause cell death of non-tumorigenic HaCaT cells. Similar results were obtained for the melanoma cell lines A-375, G-361, SK-MEL-5, MeWo and WM-115 and for the oesophageal cancer cell line KYSE-140.
Human hepatocellular carcinoma cell lines SNU-398 and SNU-449 in Minimum Essential Medium (Gibco #31095) supplemented with 10% FBS (Fetal Bovine Serum Superior, Biochrom #S0615) and 1% Penicillin/Streptomycin (PAA #P11-010) were seeded at 1 million cells/ml in 1 ml medium/well in a 6well plate. 25 μl of lentivirus expressing non-targeted or PYCR1 specific shRNAs (listed in Example 10) were added 6 hours later. The following day (day 1), the cells were washed with PBS, trypsinated and 250000 cells were seeded in 5 wells of a 6well culture plate for sample generation. Untransfected cells were used as control. On day 5, the medium was exchanged and 0.6 μg/ml puromycin was added to select for shRNA expressing cells. On day 6, lysates were generated from one well to confirm PYCR1 knockdown by Western blot and methanolic extracts were generated from 4 wells for LC-MS measurement of proline. Cells were washed twice with cold 0.9% NaCl solution and extracted with2×0.5 ml 80% cold methanol (v/v). After centrifugation, the clear supernatant was used for LC-MS/MS based proline measurement using a Waters ACQUITY UPLC I-Class system connected to a Waters Xevo TQ-S micro triple quadrupole mass spectrometer. The UPLC was equipped with an ACQUITY UPLC BEH Amide 1.7 μm 2.1×75 mm column and an ACQUITY BEH Amide 1.7 μm VANGUARD pre-column. The mass spectrometer was operating in MRM ESI positive mode. Cell pellets were lysed and protein determined (BCA Protein Assay Reagent (#23225, Thermo Scientific) for normalization. PYCR1 knockdown was confirmed and led to reduction of cellular proline levels in both cell lines (
PYCR1 knockdown results in anti-proliferative effects in these cancer cell lines.
The findings made herein do also translate to antagonists of PYCR2 (Pyrroline-5-carboxylate reductase 2, UniProtKB-Q96C36 (P5CR2_HUMAN) and PYCR3 (Pyrroline-5-carboxylate reductase 3, UniProtKB-Q53H96 (P5CR3_HUMAN)).
PYCR1 shows high sequence similarity/identity not only in its isoforms, but also with PYCR2 and PYCR3. The active sites of all three variants are well conserved, showing 100% identity in its isoforms, but still 97.1% identity between PYCR1 and PYCR2, and 85,7% similarity/60% identity between PYCR1 and PYCR3. The next-closest relative is NXRD1 (NADP-dependent oxidoreductase domain-containing protein 1), which features 32% overall identity with PYCR1 but hardly any identity/similarity in the active site.
Further, because of the sequence similarity, an involvement of PYCR2 and PYCR3 in oncogenesis, in particular of liver tumor, skin tumor and oesophagus tumor, is highly likely.
Hence, inhibitors against the active site of PYCR1, including antibodies, aptamers and small molecules, are expected to also inhibit PYCR2, while inhibition of PYCR3 appears likely at least in many cases. These inhibitors are hence likely to also exhibit an antineoplastic effect, in particular in the treatment of liver tumor, skin tumor and oesophagus tumor Similarly, nucleic acid molecules that bind to PYCR1 encoding nucleic acid may also inhibit PYCR2 and inhibition of PYCR3 appears possible.
Further, the assays disclosed herein can likewise be used to actually identify inhibitors for PYCR2 and PYCR3, e.g., by
Furthermore, proline metabolism was found to be altered in human HCC and associated with patient prognosis:
The gene expression changes found in the HCC animal models were found to be similar to changes seen in human HCC tumors in the TCGA RNA-seq data. Potent upregulation of PYCR1 and P5CS and downregulation of PRODH were observed in both analyses (
Clinical data demonstrated that patients who retained high tumor expression of PRODH, or low tumor expression of PYCR1 (and also PYCR2 and PYCR3) or P5CS had significantly better survival rates, as determined by analysis of the TGCA cohort for correlations in overall survival (
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| Number | Date | Country | Kind |
|---|---|---|---|
| 17172756.3 | May 2017 | EP | regional |
| 18165811.3 | Apr 2018 | EP | regional |
