Patent Application
20230285481
The present invention is encompassed within the field of oncology. Particularly, the present invention refers to the use of viruses belonging to the Parvoviridae family, preferably to the genus Protoparvovirus, or to combinations of such viruses with chemotherapy, in the treatment of cancer. In particular, cancer is characterized by presenting mutations in the TP53 gene and/or post-translational modifications in the p53 protein.
P53 Protein and Cancer
The p53 protein, which is expressed from the TP53 gene, performs essential functions through very diverse mechanisms in the control of the cell cycle and genomic stability, which is why it has sometimes been called “the guardian of the genome”. Therefore, it is not surprising that mutations in the TP53 gene be one of the main mechanisms responsible for induction of multiple and diverse types of cancers in humans. In fact, TP53 is the most frequently mutated gene in human cancer and, in particular, various genetic changes in the gene TP53 are frequently found in glioblastoma (GBM) (Brennan, C W et al (2013) The somatic genomic landscape of glioblastoma, Cell 155, 462-477), a devastating disease without effective treatment. The genetic alterations in TP53 and in other genes cause the growth of GBM tumors to be governed by functionally redundant signaling, which allows their adaptation in responses to directed molecular therapies. It should therefore be emphasized that, despite the enormous importance of mutations in TP53 for the initiation and progression of multiple cancers being currently suffered by millions of humans, there is no specific effective treatment against these genetic lesions, as today TP53 is mainly considered as a “non-druggable” gene (Kastenhuber, E R, and S. W. Lowe. (2017), Putting p53 in context, Cell 170.1062-1076).
Conventional Cancer Chemotherapy and p53
In the current oncology clinic, the chemotherapeutic regimes frequently use (alone or in combination) the following drugs:
Oncolytic Viruses
Multiple viruses have demonstrated anti-cancer ability (oncolysis) in different systems, used as natural strains or genetically modified. In these regards, it should be highlighted at least the following types of viruses with some demonstrated oncolytic capacity: parvovirus, measles virus, reovirus, adenovirus, herpesvirus and poxvirus. However, it has not been identified in the state of the art oncolytic viruses selectively acting against cancers harboring TP53 mutations and/or post-translational modifications in the p53 protein. In other words, the ability of oncolytic virus has not been specifically linked so far to mutations in the TP53 gene and/or post-translational modifications in the p53 protein.
The present invention therefore focuses on solving the technical problem explained above, by identifying oncolytic virus for use alone or in combination with chemotherapy, to especially target cancers with mutations in the TP53 gene and/or post-translational modifications in the p53 protein. The present invention thus provides an effective therapeutic window, allowing specific and custom treatments against tumors harboring genetic alterations in the TP53 gene and/or post-translational modifications in the p53 protein, such as those often found in human primary cancers.
The present invention refers to the use of viruses belonging to the Parvoviridae family, particularly to the Protoparvovirus genus, or combinations of such viruses with chemotherapy in the treatment of cancer. Noteworthy, many types of Cancers are characterized by presenting mutations in the TP53 gene and/or post-translational modifications in the p53 protein. Furthermore, in a preferred aspect, the present invention demonstrates that such viruses cooperate synergistically in their toxic effects against cancer cells with conventional chemotherapy, provided that the target cells harbor genetic alterations in the gene TP53 gene and/or post-translational modifications in the p53 protein, either constitutive or induced by these drugs.
Therefore, the present invention breaks a prejudice in the state of the art, because it is generally assumed a good correlation between the presence of TP53 mutations in cancer patients and the adverse outcome of chemo- and radiotherapy treatments, although the spectrum of mutations involved in each case is yet to be defined [Tchelebi, L., Ashamalla, H., and Graves P R (2014) Mutant p53 and the response to chemotherapy and radiation. In: Deb S., Deb S. (eds) Mutant p53 and MDM2 in Cancer. Subcellular Biochemistry, vol 85. Springer, Dordrecht]. In contrast, the present invention demonstrates an effective treatment of various types of cancers characterized by presenting mutations in the TP53 gene and/or a post-translational modification in the p53 protein, when the parvoviruses of the invention are used, achieving a synergistic effect in combination with genotoxic chemotherapeutic agents.
Thus, the present invention, through the figures and examples set forth below, demonstrates that:
Therefore, the first aspect of the present invention relates to a viral particle comprising a nucleotide sequence consisting essentially of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, for use, alone or in combination with genotoxic chemotherapy drugs, in the treatment of cancer, where the cancer is characterized by presenting mutations in the TP53 gene and/or post-translational modification(s) in the p53 protein. Furthermore, viral particles with at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the sequences SEQ ID NO 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 are included in the present invention.
It is important to highlight the high percentage of homology among the four viral sequences belonging to the genus Protoparvovirus of the Parvoviridae family included in the present invention, as shown in Table 1. It is noteworthy to further note that the differences in nucleotides are placed mainly in non-coding regions of the genomes, so if we stick to the coding regions the percentage of homology would be even higher.
The second aspect of the present invention refers to a pharmaceutical composition comprising a viral particle which in turn comprises a nucleotide sequence consisting essentially of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, in combination with a genotoxic chemotherapy drug.
In a preferred aspect of the invention, post-translational modifications in the p53 protein are constitutive, induced by chemotherapy drugs that induce damage to ADN or genotoxic stress [Kirkland, D. et al. Updated recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests. Mutation Research 795 (2016) 7-30] [Zhu, Y. et al. Cisplatin causes cell death via TAB1 regulation of p53 MDM2 MDMX circuitry. (2013). Genes and Develop 27: 1739-1751], or induced by oncogenic viruses. In a preferred aspect of the invention, the post-translational modification of the p53 protein consists in the phosphorylation of the Ser15 residue. In a preferred aspect of the invention, the TP53 gene mutation is selected from the group comprising: R273H, P72R, E258K, G245S and/or V173L. In a preferred aspect of the invention, the TP53 gene mutation is found in the DNA-binding domain (DBD) of the encoded p53 protein. In a preferred aspect of the invention, the genotoxic chemotherapy drug is selected from the group comprising: cisplatin, hydroxyurea, 5-fluoruracyl, gemcitabine, or cytosine arabinoside.
In a preferred aspect of the invention the cancer is glioma, lung cancer, esophageal cancer, liver cancer, pancreatic cancer, bladder cancer, colorectal cancer, prostate cancer, glioblastoma, glioma, head and neck cancer, cancer of the breast, stomach cancer, ovarian cancer, uterine cancer or melanoma. In a preferred aspect of the invention the said viral particle is used in combination with a genotoxic chemotherapy drug, where the viral particle is administered at the same time, after, or before the genotoxic chemotherapy drug.
The third aspect of the present invention refers to the in vitro method for the determination of mutations in the TP53 gene and/or post-translational modifications in the p53 protein comprising the use of a viral particle comprising a nucleotide sequence consisting essentially of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
The fourth aspect of the present invention relates to a method for the in vitro diagnosis of cancer, or for selection of cancer patients comprising the determination of mutations in the TP53 gene and/or post-translational modifications in the p53 protein by using the viral particle comprising a nucleotide sequence consisting essentially of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
The fifth aspect of the present invention relates to mutations in the TP53 gene and/or post-translational modifications in the p53 protein for use in the treatment of cancer, where the mutation of TP53 is selected from the group comprising: R273H, P72R, E258K, G245S or V173 L, and the post-translational modification of the p53 protein is phosphorylation at the Ser15 residue.
The sixth aspect of the present invention refers to a method for the treatment of cancer, characterized by presenting mutations in the TP53 gene and/or post-translational modifications in the p53 protein, comprising the administration of a therapeutically effective amount of a viral particle comprising a nucleotide sequence consisting essentially of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and/or chemotherapy.
In a preferred aspect of the present invention the mutation of the TP53 gene is selected from the group comprising: R273H, P72R, E258K, G245S and/or V173 L. In a preferred aspect of the present invention the mutation of the TP53 gene is found in the DBD region or in the proline-rich domain (PRD) of the encoded p53 protein. In this regard, it is important to keep in mind that most mutations, and many of the more important mutations that occur in the TP53 gene, are present in the domain DBD gene, and also that this fact may be considered reproducible in different types of cancers, as seen in
Preferably, in the said pharmaceutical composition or medicament, the viral particle is in a concentration of between 106 to 1012 pfu/ml (pfu: plaque forming unit), more preferably between 107 and 1011 pfu/ml, even more preferably between 108 and 1010 pfu/ml.
This concentration of viral particles in the pharmaceutical composition can be referred, within the framework of the invention, to the concentration of a single type of viral particle, the pharmaceutical composition not containing any other type of viral particle.
Alternatively, the indicated concentration can be achieved by mixtures of various types of viral particles as defined above, together reaching the indicated concentration. All possible combinations that will look apparent to one skilled in the art are included within the scope of the present invention.
The pfu/ml is a quantitative measure usually used in virology, and corresponds to the number of infectious viral particles capable of forming lysis plaques in monolayers of susceptible cells per volumetric unit. It is a functional measure rather than a measure for the absolute number of particles: virus particles that are defective or that fail to infect their target cells will not produce a plaque, and therefore will not be counted. For example, a composition comprising parvovirus MVM in a concentration of 106 pfu/ml indicates that 1 milliliter of the composition contains enough virus particles to produce 106 lysis plaques in a cell monolayer, but it is not possible to establish a relationship between pfu and the actual number of physical virus particles by this assay. By complementary methods, for example by agglutination of red cells or by electron microscopy, it is possible to determine the total number of viral particles in a preparation whether or not infectious.
According to a preferred embodiment, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.
According to a preferred embodiment, the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient According to another preferred embodiment, the pharmaceutical composition comprises at least one pharmaceutically acceptable adjuvant.
Preferably, in the pharmaceutical composition the vehicle or excipient is such as to allow administration of said composition intratumorally (in solid tumors), intracerebrally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intracutaneously, intracecally (or intrathecally), intraventricularly, orally, enterally, parenteral, intranasal or dermal. More preferably, the administration is intracerebrally, intravenously, or intranasally.
In a particularly preferred embodiment the present invention refers to: A viral particle comprising a nucleotide sequence consisting essentially of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, for use in the treatment of cancer, wherein the cancer is characterized by presenting at least one mutation selected from the group comprising or consisting of: mutation R273H in the gene TP53, mutation P72R in the gene TP53, mutation E258K in the gene TP53, mutation G245S in the gene TP53, mutation V173L in the gene TP53, and/or phosphorylation of the p53 protein.
In a preferred embodiment, the phosphorylation of p53 protein is constitutive, or it has been previously induced by genotoxic chemotherapy drugs or by oncogenic viruses.
In a preferred embodiment, the phosphorylation of p53 protein consists of the phosphorylation of the Ser15 residue.
In a preferred embodiment, the mutation is placed in the DBD region of the TP53 gene or in the PRD region of the p53 protein.
In a preferred embodiment, the genotoxic chemotherapy drug that induces the phosphorylation in p53 protein is selected from the group comprising: cisplatin, hydroxyurea, 5-fluoruracyl, gemcitabine or cytosine arabinoside.
In a preferred embodiment, the cancer is selected from the group comprising: glioma, glioblastoma, acute myeloid leukemia, lung adenocarcinoma, bladder carcinoma or rectal adenocarcinoma, which present the R273H, P72R, E258K, G245S and/or V173L mutations in the TP53 gene.
In a preferred embodiment, the viral particle comprising a nucleotide sequence consisting essentially of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 is used in combination with one genotoxic chemotherapy drug, wherein the viral particle is administered after the chemotherapy drug.
In a preferred embodiment, the cancer is characterized by presenting the R273H mutation in the TP53 gene, selected from the group comprising: lung cancer, cancer esophagus, liver cancer, pancreatic cancer, bladder cancer, colorectal cancer, prostate cancer, glioblastoma, glioma, head and neck cancer, breast cancer, stomach cancer, ovarian cancer, cancer of the uterus, or melanoma.
In a preferred embodiment the genotoxic chemotherapy drug is selected from the group comprising: cisplatin, hydroxyurea, 5-fluoroacyl, gemcitabine or cytosine arabinoside.
In a preferred embodiment, the present invention refers to a pharmaceutical composition comprising a viral particle which in turn comprises a nucleotide sequence consisting essentially of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 in combination with a genotoxic chemotherapy drug, for use in the treatment of cancer, wherein the viral particle is administered after the chemotherapy drug.
In a preferred embodiment the present invention refers to a method for treating a cancer type characterized by presenting at least one mutation selected from the group comprising or consisting of mutation R273H in the gene TP53, mutation P72R in the gene TP53, mutation E258K in the gene TP53, mutation G245S in the gene TP53, mutation V173L in the gene TP53, and/or phosphorylation of the p53 protein. This method comprises the administration of a pharmaceutical composition comprising a viral particle which in turn comprises a nucleotide sequence consisting essentially of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, which, in a preferred embodiment is combined with a genotoxic chemotherapy drug.
For the purposes of the present invention, the following definitions are provided:
The human glioblastoma stem cells were obtained from tumor explants provided by the Neurosurgery service of the Ramón y Cajal Hospital in Madrid. Explants were diagnosed as glioblastoma grade IV by histochemistry performed by the Pathology service of the same hospital. In all cases, the informed consent of the patients was obtained and the approval of the Institutional Ethics Committee of the Ram6n y Cajal Hospital in Madrid. Further research in tissue culture was authorized by the respective Ethics Committee of the Universidad de Madrid, and Centro de Biologiá Molecular Severo Ochoa (CSIC-UAM). The biopsies, collected at the foot of the operating room, were mechanically and enzymatically disintegrated, and finally the cell suspension was filtered to be cultivated in the DMEM medium: F12 (1:1) supplemented with various factors.
On the other hand, the established cell lines of mouse, human cancers and other mammals were cultured in Dulbecco's Modified Eagle medium (DMEM) buffered with 0.3% NaHCO, and in atmosphere of 5% CO2. The medium was supplemented with antibiotics (75 U/ml streptomycin, 75 μg/ml penicillin G), 2 mM L-Glutamine and 5% of fetal bovine serum (FCS) des-complemented at 56° C. for 30 minutes. The following cell lines have been used:
In all the experiments, a minimum of 30,000 events per sample were acquired in a FACSCantoII cytometer (BD Biosciences), which were pre-selected by a region based on their size and complexity (FSC and SSC parameters, respectively) in order to exclude cellular debris and other contaminating particles. The CellQuest software (BD Biosciences) was used to acquire the events and the FlowJo software (Tree Star) was used to analyze the data. The cell cycle analysis was in accordance to [Gil-Ranedo, J., Hernando, E., Riolobos, L., Dominguez, C., Kann, M, and José M Almendral. 2015. The mammalian cell cycle regulates nuclear parvovirus capsid assembly. PlosPathogens, 11; 11 (6): e1004920]. For staining, cells were permeabilized with PBS+0.1% triton X-100 for 10 minutes at room temperature, then blocked with the same buffer supplemented with 1% FCS for 20 minutes. Cells were re-suspended in PBS (pH 7.2)+0.5% BSA and the primary antibodies indicated in the figures were added followed by 1 h stirring at 37° C. Cells were washed in PBS and the secondary antibodies were added and incubated similarly. Two further washes in PBS were made before analyzing the samples in the flow cytometer.
In the purification of cells by flow cytometry (FACS) for the genetic analysis of TP53 (as shown in the
The clonogenic capacity and cell viability were estimated by a colony formation assay based on [Rubio, M P, Guerra, S., and Almendral, J M 2001. Genome replication and postencapsidation functions mapping to the nonstructural gene restrict the host range of a murine parvovirus in human cells. J Virol 75 (23): 11573-11582].
Immunofluorescence analyses were performed on neurospheres of hGSCs (human glioblastoma stem cells) and established adherent cell lines as follows:
MVM belongs to the family Parvoviridae, genus Protoparvovirus. The prototypic strain of this virus (MVMp) was originally isolated from fibroblasts [Crawford, L. (1966) A minute virus of mice, Virology, 29, p. 605-612] and the so-called immunosuppressive strain (MVMi) from mouse lymphocytes [Bonnard, G D, Manders, E K, Campbell, D A, Herberman, R B and Collins, M J (1976) Immunosuppressive activity of a subline of the mouse EL-4 Lymphoma, J Exp Med, 143 (1), pp. 187-205. DOI: 10.1084 jem.143.1.187]. Only the MVMi strain is pathogenic in mice.
The preparations of the parvovirus MVM (p, i) were obtained in NB324K cells following established protocols [Segovia, J C, Gallego, J M, Bueren, J A and Almendral, J M (1999) Severe leukopenia and dysregulated erythropoiesis in SCID mice persistently infected with the parvovirus minute virus of mice., Journal of Virology, 73 (3), pp. 1774-84], [Sánchez-Martínez, C., Grueso, E., Carroll, M, Rommelaere, J. and Almendral, J M (2012) Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry. Virology, 432 (1), pp. 45-56], [Gil-Ranedo, J., Hernando, E., Valle, N., Riolobos, L., Maroto, B. and Almendral, J M (2018) Differential phosphorylation and n-terminal configuration of capsid subunits in parvovirus assembly and viral trafficking, Virology, 518, pp. 184-194]. For this, we used plasmids containing the complete genome of MVMp and MVMi strains, including the palindromic sequences of the [Gardiner, M S and Tattersall, P. 1988. Mapping of the fibrotropic and lymphotropic host range determinants of the parvovirus minute virus of mice. J Virol 62 (8): 2605-2613] [Hirt, B., Colomar, M C and Beard, P. (1998) Two segments in the genome of the immunosuppressive minute virus of mice determine the host-cell specificity, viral control DNA replication and affect viral RNA metabolism, Journal of General Virology, 79, 581-586] which allows obtaining viral particles by transfection of cells [Sánchez-Martínez, C., Grueso, E., Carroll, M., Rommelaere, J. and Almendral, J M (2012) Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry. Virology, 432 (1), pp. 45-56]. Using virus directly produced by transfection (48 h post-transfection), NB324K cells grown to confluence in ten P100 plates (a total of 5×107 cells) were infected at a multiplicity of infection (MOI) of 0.005 plaque-forming units/cell (PFU/cell) in 1 ml of complete PBS (PBSc; PBS) with 0.9 mM CaCl2 and 0.5 mM MgCl2) with 0.1% FCS. After 1 h at 37° C., the inoculum was removed and incubated in DMEM with 5% FCS for 5 h to allow internalization of the virus. Subsequently the adhered cells were detached with trypsin-EDTA, diluted in 400 ml of DMEM with 5% FCS and seeded onto fifty P100 plates. Cells were incubated until the appearance of cytopathic effect (approximately 5 days).
We followed protocols described by [Santarén, J F, Ramírez, J C and Almendral, J M (1993) Protein species of the parvovirus minute virus of MVMp strain: involvement of phosphorylated VP-2 subtypes in viral morphogenesis., Journal of Virology, 67 (9), pp. 5126-38], [Hernando, E., Llamas-Saiz, A L, Foces-Foces, C., McKenna, R., Portman, L, Agbandje-McKenna, M. and Almendral, J M (2000) Biochemical and Physical Characterization of Parvovirus Minute Virus of Mice Virus-like Particles, Virology, 267 (2), pp. 299-309], [Sánchez-Martínez, C., Grueso, E., Carroll, M., Rommelaere, J. and Almendral, J M (2012) Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry. Virology, 432 (1), pp. 45-56] and [Gil-Ranedo, J., Hernando, E., Riolobos, L., Dominguez, C., Kann, M., and José M. Almendral. 2015. The mammalian cell cycle regulates nuclear parvovirus capsid assembly. PlosPathogens, 11; 11 (6): e1004920]. In short, the virus present in the medium was recovered by precipitation with 3.4% polyethylene glycol 6000 and 0.5 M NaCl overnight at 4° C. and subsequently centrifuged at 5000 rpm for 30 minutes in an angular Sorvall GSA rotor. To recover the intracellular virus, the cell pellet was resuspended in 50 mM Tris-HCl pH 7.5, 1 mM EDTA (TE) and subjected to three consecutive freeze/thaw cycles, after which 0.2% SDS was added and clarified at 8000 rpm, 10 min at 4° C. in a Sorvall HB4 swinging rotor. The virus recovered from the medium and the intracellular virus were pooled and centrifuged through a 20% sucrose cushion (Merck) in 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.1 M NaCl and 0.2% SDS for 18 h at 16000 rpm in a TST 28.38 rotor. The pellet was resuspended in TE with 0.2% Sarkosyl (Sigma) and banded to equilibrium in a CsCl gradient (ni=1.371) run for 24 h at 50000 rpm and 15° C. in a TFT 80.13 rotor. Fractions of 0.5 ml were collected by syringe from the top of the gradients and the presence of empty capsids and DNA-filled viruses was determined by hemagglutination with mouse erythrocytes. Finally, the fractions containing the viruses were pooled and dialyzed against PBS. Purified virus was stored in aliquots at −70° C.
This method was used to estimate the amount of virus particles and follow up purifications. It was based on [Sánchez-Martínez, C., Grueso, E., Carroll, M, Rommelaere, J. and Almendral, J M (2012) Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry. Virology, 432 (1), pp. 45-56] and [Hernando, E., Llamas-Saiz, A L, Foces-Foces, C., McKenna, R., Portman, L, Agbandje-McKenna, M. and Almendral, J M (2000) Biochemical and Physical Characterization of Parvovirus Minute Virus of Mice Virus-like Particles, Virology, 267 (2), pp. 299-309]. For the hemagglutination (HA) assay, adult mouse blood was washed three times with phosphate saline (PBS), collecting the erythrocytes by centrifugation in a tabletop centrifuge at 1500 rpm for 5 min. After several washes the erythrocyte sediment was resuspended as 50% (v/v) in PBS and kept at 4° C. until use. The HA was carried out in U-profile microtest plates (Nunc). The samples to be evaluated were applied in a final volume of 100 μl in PBS and serial dilutions 1:2 in PBS were made. Finally, 50 μl of 2% erythrocytes in PBS was added to each well, the plate was gently shaken and kept at 4° C. in darkness for at least two hours. The title was obtained from the inverse of the highest dilution that maintains the hemagglutinating capacity.
Based on [Gil-Ranedo, J., Hernando, E., Valle, N., Riolobos, L., Maroto, B. and Almendral, J M (2018) Differential phosphorylation and n-terminal configuration of capsid subunits in parvovirus assembly and viral trafficking, Virology, 518, pp. 184-194]. NB324K cells seeded 24 h before in P60 plates at a density of 2.2×10e5 cells/plate were used. The culture medium was removed, cells washed in PBS with Ca++ and Mg++(complete or PBSc), and the viral inoculum was added in 400 l per P60 diluted in PBSc supplemented with 0.1% FCS. After one-hour adsorption at 37° C. with gentle agitation, the inoculum was removed and 7 ml of plating medium (DMEM, 10% FCS, and 0.6% agarose LM-GQT Pronadisa) equilibrated at 37° C. was added. After a 6-days incubation, the plates were fixed in 10% formaldehyde (Panreac) in PBS and stained with 0.2% violet crystal (Panreac) in 10% formaldehyde prepared in PBS. The count of the number of lysis plaques multiplied by the dilution allows to obtain the infectious titer of the virus in plaque forming units per milliliter (PFU/ml).
The low molecular weight DNA from cells electroporated with plasmids, or infected with MVM, was obtained by a modified Hirt's method [Segovia, J C, Gallego, J M, Bueren, J A and Almendral, J M (1999) Severe leukopenia and dysregulated erythropoiesis in SCID mice persistently infected with the parvovirus minute virus of mice., Journal of Virology, 73 (3), pp. 1774-1784]. Briefly, the transfected cells were lysed in Hirt's solution (50 mM Tris pH 7.5, 0.5% SDS, 10 mM EDTA) supplemented with 20 μg/ml tRNA carrier to ensure recovery, and digested with proteinase K (100 μg/ml) (Merck) for 2 hours at 37° C. The reaction was adjusted to 1M NaCl and the genomic DNA was precipitated overnight at 4° C. The enriched fraction of low molecular weight viral DNA was obtained from the supernatant after centrifuging the samples at 4° C. and 14 K rpm for 30 minutes in a microfuge (Eppendorff). This DNA was precipitated with 0.3 M NaCl and 2.5 volumes of absolute ethanol at −20° C., washed with 70% ethanol to remove salts, and resuspended in water or in 50 mM Tris pH 7.5 and 1 mM EDTA.
Samples were electrophoresed 2-3 h at 60V on a 0.8% agarose gel (Gibco) in Tris-Borate-EDTA buffer (45 mM Tris-borate, 1 mM EDTA) with 5 μg/ml ethidium bromide (Boehringer) together with molecular weight markers (HindIII digest of phage 029 DNA) and controls of replicative forms and ssDNA of MVM. The transfer was made to a Nylon membrane (Hybond-N+, Amersham, Pharmacia) in 0.4 M NaOH overnight. Finally, the membrane was washed in a 2×SSC solution for 10 minutes at room temperature (20×SSC is: 3 M NaCl, 0.3 M sodium citrate) and dried 2 hours at 78° C.
The membrane was incubated in pre-hybridization solution (5×SSC, 5×Denhardts' solution [Ficoll (Ty400), Polyvinylpyrrolidone, BSA], 10 mM Tris-HCl pH 7.5, 0.5% SDS, 50% Formamide), to eliminate possible nonspecific binding, for four hours at 42° C. Next, the solution was replaced by the hybridization solution, which is formed by the same components of the pre-hybridization solution together with the denatured probe. The probe was the full-length the MVM genome labeled in vitro to high specific activity with 32P by “random priming” generally using dCTP-alpha 32P and purified by a Sephadex G-50 spin-column. Hybridization was allowed at 42° C. for one or two days, and finally membranes were washed with a solution of 0.1×SSC and 0.5% SDS at 50° C. for three hours, before exposure to X-ray films.
The primary and secondary antibodies used in immunological techniques were:
Hydroxyurea was obtained from Calbiochem (Hydroxyurea cat 400046-5 gm). 5-Fluoruracil (5FU) was obtained from Sigma (Ref F-6627-1G). Cisplatin from EMC Millipore (232120-50 mg).
samples, once denatured by boiling for five minutes in loading buffer (10% glycerol (Merck), 5% β-mercaptoethanol (Merck), 0.002% bromophenol blue (Merck), 0.5M Tris-HCl pH 6.3, 0.4% SDS), were resolved by denaturing gel electrophoresis of 8% polyacrylamide. Electrophoresis was carried out in a Tris-Glycine buffer (25 mM Tri-HCl (Serva), 192 mM Glycine (Gibco), 0.1% SDS) for two-four hours at 100 V in minigels (10×10×0.1 cm), with molecular mass markers run in parallel (“Prestained SDS-PAGE Standards, Broad Range” (Biorad), or “Protein Molecular Weight Standards, Broad Range”, Amersham). The samples were transferred to nitrocellulose memebrane (Schleicher and Schuell) in transfer buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS, 20% methanol) for one hour at 100 V (Trans-blot electrophoretic transfer Cell, Biorad).
The membrane was hydrated in TBS-T buffer (20 mM Tris pH 7.5, 140 mM NaCl, 0.1% Tween 20) and incubated under shaking for one hour at 4° C. in TBS-T with 10% fetal bovine serum (FBS). After washing with TBS-T it was incubated with the primary antibody diluted in TBS-T with 1% FBS and 1% NP40, for 24 h at 4° C. After thorough washing, the secondary antibody was added at incubated for 1 h at RT. Finally, the membrane was washed with TBS-T and TBS (without Tween 20), revealed with the ECL system (“Enhanced Chemiluminiscence”, Amersham) and exposed to autoradiograpy (Kodak).
To express the p53 protein in cell cultures the pCMV-neo-p53 plasmid was used [Baker S J, Markowitz S, Fearon E R, Willson J K, and Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 1990, 249 (4971): 912-5], kindly provided by J. Paramio (Ciemat, Madrid). The plasmid named “Helper” and the plasmid to express the E4orf6 oncogene of Adenovirus [Winter, K., von Kietzell, K., Heilbronn, R., Pozzuto, T., Fechner, H., and S. Weger. (2012). Roles of E4orf6 and VA I RNA inadenovirus-mediesated stimulation of human parvovirus B19 DNA 30 replication and structural gene expression. J. Virol. 86, 5099-5109] were kindly donated by S. Weger (Charité, Berlin). Plasmids were transfected into cells by a chemical (Jetpaid) or physical (electroporation) method depending on cell type.
Cell samples were processed to obtain total RNA using Trizol and conventional protocols. The RNA was then copied to cDNA using Reverse Transcriptase and random primers. The thus obtained cDNA was used to sequence the human or mouse TP53 gene using the TP53 sequence and primers listed in the Annex. Methods followed were the conventional Sanger sequencing, or massive sequencing (NGS) for the RNA samples obtained from FACS-sorted GSs (see
We next studied whether the DDR mounted by the GS in response to the parvovirus MVM involves, in a cell-type and/or virus-strain dependent, the modification at the Serine 15 residue of p53 by phosphorylation (Pp53-S15). In uninfected GS neurospheres, a basal expression of Pp53-S15 was observed, with some prominent cells in GS5 and GS7, but when infected by MVMp the Pp53-S15 is clearly induced in a significant fraction of the cells expressing NS1 (
To extend the previous study to other types of cancers, as well as to the cells used to grow these viruses, several human and other mammals cell lines were infected with the two virus strains (MVMp, MVMi) and analyzed for the NS1 expression and virus genome replications, in relation to the presence of mutations in the TP53 gene and to post-translational modifications of the p53 protein. The results obtained in these analyses were:
To analyze the marked difference between the NS1+ cells allowing or not vDNA synthesis, we proceeded to sort these two cell populations and to characterize their TP53 genetic. Sorted A9 NS1+ cells behaved again distributed in two populations vDNA+/− of similar size (
Although the correlation between genetic alterations in TP53 and/or modifications in the p53 protein with the expression of the NS1 protein of MVM was consistent in several transformed cell lines (
The analyses with established lines supported the association found between the Pp53-S15 modification and the permissiveness to MVM infection in GS cells (
In summary, the following relevant conclusions can be drawn from this analysis:
The high permissiveness to NS1 expression and MVM genome replication in cancer lines with constitutive Pp53-S15 staining and expressing viral oncogenes (
As MVM infection correlated with Pp53-S15 expression (
Consequently, the CisPt dose effect on different MVMp life cycle parameters, and on p53 phosphorylation and functional signaling in U373-MG cells, was analyzed. For this, the U373-MG cells treated with Cis-Platinum doses (10-120 microM) for 1 hour at 37° C. were inoculated with MVMp to allow adsorption, and then of the infection was allowed to progress for 24 hours. Cells were sampled and processed for virus macromolecular markers and Pp53-S15 determinations. A significant increase in the Pp53-S15 levels was observed by IF-confocal across all the assayed 5-20 microM range of Cis-Pt doses (
We finally analyzed whether these MVM/CisPt cooperative effects impact cancer cells viability, and therefore whether they imply a therapeutic significance. As seen in
In a second study, this analysis of CD/MVM combined therapy was extended to the U87-MG human glioblastoma cell line, which lacks TP53 mutations but expressed post-translationally modified p53 protein forms (
In a second analysis the CD/MVMp cooperation was analyzed. MVMp infection of U87-MG (
| Number | Date | Country | Kind |
|---|---|---|---|
| 18382956.3 | Dec 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/086048 | 12/18/2019 | WO |
