WO2019246376A1 - Prévention du cancer primaire et résistant au traitement par des inhibiteurs de la transcriptase inverse endogène - Google Patents

Prévention du cancer primaire et résistant au traitement par des inhibiteurs de la transcriptase inverse endogène Download PDF

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WO2019246376A1
WO2019246376A1 PCT/US2019/038205 US2019038205W WO2019246376A1 WO 2019246376 A1 WO2019246376 A1 WO 2019246376A1 US 2019038205 W US2019038205 W US 2019038205W WO 2019246376 A1 WO2019246376 A1 WO 2019246376A1
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cancer
inhibitor
patient
cells
diagnosed
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Olga B. CHERNOVA
Andrei Gudkov
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • 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
    • C12Q2600/158—Expression 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)
    • G01N2333/9125—Nucleotidyltransferases (2.7.7) with a definite EC number (2.7.7.-)
    • G01N2333/9128—RNA-directed DNA polymerases, e.g. RT (2.7.7.49)

Definitions

  • the present invention relates to the prevention of cancer or prevention of relapse of cancer in a patient via administration of an RT inhibitor.
  • Cancer is a heterogeneous disease. It manifests in a wide variety of tissue sites, with different degrees of dedifferentiation, invasiveness, and aggressiveness. Cancer cells represent dedifferentiated cells which have regained features strongly resembling stem cells, such as limitless proliferation potential and non-specific cell lineage morphology. Some of the characteristics that cancer cells adopt are the reactivation of endogenous reverse transcriptase (RT), which is originally highly expressed in embryonic and poorly differentiated cells.
  • RT reverse transcriptase
  • HIV-l Human immunodeficiency virus type 1 (HIV-l) reverse transcriptase inhibitors (RTIs) are widely used in the clinical treatment of HIV/AIDS. Apart from their use in the treatment of HIV/AIDS, certain RTIs also exhibit anti-cancer activity.
  • HIV-l Human immunodeficiency virus type 1
  • RTIs also exhibit anti-cancer activity.
  • a reverse transcriptase (RT) inhibitor is administered to a patient.
  • a method of preventing a primary cancer in a patient wherein the method includes identifying a patient that does not have HIV and that has never been diagnosed with any cancer and administering to said patient an RT inhibitor.
  • a method of preventing a primary cancer in a patient wherein the method includes identifying a patient that is diagnosed as having a genetic predisposition to cancer, but who has never been diagnosed with any cancer; and administering to said patient an RT inhibitor.
  • a method of preventing a primary cancer in a patient includes identifying a patient that is diagnosed as having a genetic predisposition to cancer, but who has never been diagnosed with any cancer; and administering to said patient an RT inhibitor.
  • the patient does not have HIV.
  • the patient is identified and diagnosed as having a mutation in the BRCA1 or BRCA2 gene leading to an increased risk of cancer.
  • the patient is identified and diagnosed as having a mutation in p53 gene leading to increased risk of cancer.
  • the patient is identified and diagnosed as having a mutation in, or increased expression of, or decreased expression of a target gene on Table 2 shown below leading to increased risk of cancer.
  • the patient is identified and diagnosed as having a mutation/hypermethylation (leading to under expression) in the DNA mismatch repair (MMR) system affecting genes MLH1, MSH2, MSH6 and PMS2.
  • MMR DNA mismatch repair
  • a method of preventing a primary cancer in a patient or cancer relapse in a patient comprising, identifying a patient that is diagnosed as having a genetic predisposition to cancer and/or who has been diagnosed with cancer; and administering to said patient an RT inhibitor and one or more anti-cancer drugs on Table 1 or 2.
  • the one or more anti-cancer drugs of Table 1 or 2 is administered prior to the RT inhibitor.
  • the one or more anti-cancer drugs of Table 1 or 2 is administered concurrent with the RT inhibitor.
  • RT inhibitor such that the activity of an RT encoded by ORF2 of LINE 1 (wherein the RT is referred to as RT line ) in the patient and/or cells is inhibited, and whereby the cells exhibit reduced genetic instability subsequent to the administration.
  • Genetic instability includes but is not necessarily limited to chromosomal insertions, amplifications/deletions and point mutations that are driven at least in part by RT LINE , which is not believed to be expressed in normal cells.
  • the disclosure includes suppression of genomic instability in somatic cell populations by either inhibiting RT LINE activity with drug agents, or stimulating the immune system to eradicate cells with activated functional LINE elements. Accordingly, the disclosure is may be pertinent to uses in cancer cells, pre-cancerous cells, somatic cells, and combinations thereof.
  • the RT inhibitor can be introduced into progeny of the population of the cells, thereby obtaining progeny of the population of cells that comprise reduced genetic instability.
  • use of an RT inhibitor as described herein results in at least one of: fewer point mutations, fewer chromosomal insertions, fewer chromosomal deletions, fewer amplifications, a reduction in metastatic capability, a reduction in immortalization capacity, or a reduction in development to resistance to one or more pharmaceutical agents, wherein the reduction in genetic instability is relative to a control.
  • the disclosure relates to suppression of point mutations that are caused at least in in part by endonuclease activity of an integrase component of the RT; and/or to insertions that comprise integration of new copies of one or more repeat elements, or integration of new pseudogenes, or a combination thereof; and/or to deletions that comprise loss of a segment of a chromosome that is surrounded by integrated copies of repeat elements; and/or to genetic amplifications that comprise new copies of genome fragments that contain one or more functional genes surrounded by newly integrated copies of repeat elements.
  • the disclosure relates to RT inhibitor administration to a patient and/or population of cells such that at least some cells in the patient or population of cells and/or progeny of said cells exhibit at least one of: i) less resistance to a pharmaceutical agent, and/or ii) reduced metastatic capability, or iii) less evidence of aging, relative to a control.
  • the patient and/or population of cells is not infected by a human immunodeficiency virus (HIV), or other retrovirus, and the cells in the patient or population do not comprise an integrated HIV provirus, or another integrated provirus of a retroviral origin.
  • HIV human immunodeficiency virus
  • the RT inhibitor may be a nucleoside analog RT inhibitor, or nucleotide analog RT inhibitor, or a combination thereof.
  • suitable RT inhibitors include stavudine and lamivudine, and additional examples of suitable RT inhibitors are described below.
  • Figure 1 provides, without wishing to be bound by theory, a schematic description of a new paradigm for anticancer and antiaging treatments based on pharmacological and immunotherapeutic targeting of cells expressing reverse transcriptase encoded by LINE1 family of elements (LINE1 RT).
  • Figure 2 depicts a work-flow for a LINE1 retrotransposition assay as described in Moran et al. Cell. 1996 87(5):9l7-27 (shown in box to the left).
  • HeLa cells were transfected with the reporter plasmid; appearance of neo-resistant colonies is indicative of retrotransposition events; the top plate images show stavudine is effective at concentrations above 5mM. Lower panel demonstrates lack of effect of the indicated concentrations of stavudine on clonogenicity of HeLa cells.
  • Figure 3 provides a graphical summary (left panel) and plate assays image (right panel) results obtained on from use of representative RT inhibitors stavudine (STV) and azidothymidine (AZT), and demonstrates suppress acquisition of drug resistant cell variants in a population of colorectal cancer cells HCT116.
  • Figure 4 provides a graphical summary of results demonstrating that the representative HIV RT inhibitor stavudine prolongs tumor-free survival of p53-null C57BL6/j male mice.
  • Figure 5 provides a graphical summary of results demonstrating the representative RT inhibitor stavudine delays occurrence and reduces incidence of tumor relapses following initial response to standard chemotherapy in mouse spontaneous neuroblastoma model.
  • Figure 6 provides a graphical summary of tumor incidence in a transgenic mouse spontaneous breast cancer model (MMTV-neuT) following STV treatment.
  • Mammary pads were palpated every other day for detection of newly arisen tumor masses.
  • the incidence of tumor-free mice was monitored throughout the lifespan of the mice (experiment ongoing, mice currently 16 months of age), with tumors appearance starting at 10 months of age.
  • Figure 7 provides a graphical summary of the effect of chronic STV treatment on tumor development of resistance to 17-DMAG.
  • Mice from 2 tumor bearing groups were treated with 4 weekly IP injections of 17-DMAG 20mg/kg until tumors disappeared.
  • mice were on drug until the complete response and after this until the time of tumor relapse.
  • Time of tumor relapse (weeks) was marked for each mouse.
  • the animals started to receive STV with drinking water and 17-DMAG treatment as in previous group. All animals in this group were on double drugs treatment for the period until tumor relapsed. Time from tumor complete response until relapse (weeks) was marked for each mouse.
  • the present disclosure relates to prevention of a primary cancer and/or prevention of cancer relapse in a patient by administering an RT inhibitor to the patient.
  • a patient of the methods described herein is administered an RT inhibitor and an anti-cancer drug, for example as shown on Table 1 and/or Table 2 below or described elsewhere herein.
  • a patient of the methods disclosed herein has, had or is at risk of having a cancer, for example a cancer listed on Table 1 or Table 2 or described elsewhere herein.
  • a patent of the methods described herein has a mutation in, or increased expression of, or decreased expression of a target gene, for example a target gene listed in Table 2 or described elsewhere herein.
  • Additional anti-cancer therapies include one or more of radiotherapy, hormonal, tyrosine kinase inhibitor, anthracy cline, alkylating agent, topoisomerase inhibitor, antimetabolites/cytotoxic drug, BRAF inhibitor, antitumor antibiotic, isoquinololine alkaloid, Bcl-2 inhibitor, hematopoietic cell transplantation (HCT), telomerase inhibitor, nucleoside analogue reverse-transcriptase inhibitor, DNA cross-linking agent, ribonucleotide reductase inhibitor, microtubule inhibitor, and miRNA.
  • radiotherapy hormonal, tyrosine kinase inhibitor, anthracy cline, alkylating agent, topoisomerase inhibitor, antimetabolites/cytotoxic drug, BRAF inhibitor, antitumor antibiotic, isoquinololine alkaloid, Bcl-2 inhibitor, hematopoietic cell transplantation (HCT), telomerase inhibitor, nucleoside analogue reverse-tran
  • the present disclosure in various embodiments may relate at least in part to the concept that transcriptional activation of retroelements can occur either spontaneously or following specific stresses (i.e., genotoxic conditions) in rare somatic cells of mammalian organisms.
  • Figure 1 provides certain background and/or basis to some of the embodiments of the present disclosure and some embodiments and aspects disclosed herein may be based on improvements or developments of the concepts of Figure 1. Without intending to be constrained by any particular theory, it is considered that“desilencing” results in expression of endogenous reverse transcriptase (RT) encoded by LINE1 elements, which drives synthesis of cDNA copies of a variety of RNA (i.e..
  • RT reverse transcriptase
  • LINE1 RNA RNA of SINE elements, protein-coding mRNAs, tRNAs, small nuclear RNAs, etc.
  • integration into random sites of cellular DNA This leads to insertional mutagenesis associated with gene inactivation or synthesis of aberrant products (integration into open reading frames), modulation of expression (integration into regulatory regions), and gene amplification (when similar repeats are integrated around certain gene facilitating homologous recombination), among others.
  • RT of LINE1 generates DNA breaks via its endonuclease activity thereby inducing point mutations.
  • the disclosure is based at least in part on the approach that, because all these processes depend at least in part on the endogenous RT of LINE1 elements - they can be inhibited and/or prevented by using targeting the RT and/or cells that express this RT.
  • the disclosure relates to inhibition and/or prevention of cancer, aging and age-related diseases and for suppression of cancer progression.
  • the disclosure comprises methods for reducing the occurrence of genetically altered cells, such as cells with elevated genomic instability, phenotypic plasticity and adaptability, and other features that are described more fully herein.
  • the disclosure pertains to reducing such genetically altered cells in a population of cells such as cancer cells in a patient.
  • methods of this disclosure comprise exposing such cells and/or a patient to one or more inhibitors of RT and/or vaccination against such cells.
  • a gene expression signature of a cancer in a human sample is useful for assessing a patient’s cancer risk.
  • a gene expression signature of a cancer may be indicative of a patient’s risk level for a cancer and could indicate that the patient has a poor prognosis for survival with conventional therapy.
  • the prognosis may be an expected (e.g., greater than about 50%, or about 60%, or about 70%, or about 80%, or about 90% chance) survival of less than about five years, less than about three years, less than about two years, or less than about one year.
  • the prognosis and/or risk assessment may be based on the type of cancer, including population response rates of the cancer type to radiotherapy and/or chemotherapy and/or may be based upon a molecular characterization of the tumor cells, including gene expression levels of, for example, VEGF, PDGFR-b, CD31, HER2, PTEN, ERCC1, BRCA1, TOP02a, Ki-67, P53, TS, ER, PR, or mutations in one or more of EGFR, ALK, KRAS, BRAF, and PI3K.
  • cancer risk can be assessed by a gene expression signature of the cancer that is indicative of chemotherapy resistance, likelihood of cancer recurrence, or a high risk group for survival.
  • Gene expression signatures are becoming increasingly available for predicting tumor response to therapy and/or other classification of tumors for prognosis. Exemplary gene expression signatures are described in PCT/US2012/022594 (colon cancer), U.S. Patent No. 8,211,643 (NSCLC), U.S. Patent Publication No. 2010-0331210 (breast cancer), U.S. Patent No. 7,056,674, U.S. Patent No. 7,081,340, U.S. Patent No. 7,569,345, and U.S. Patent No. 7,526,387, each of which is hereby incorporated by reference in its entirety.
  • the present invention includes the measurement of a tumor specimen, including biopsy or surgical specimen samples.
  • the biopsy is a human biopsy.
  • the biopsy is any one of a frozen tumor tissue specimen, cultured cells, circulating tumor cells, and a formalin-fixed paraffin-embedded tumor tissue specimen.
  • the tumor specimen may be a biopsy sample, such as a frozen tumor tissue (cryosection) specimen.
  • a cryosection may employ a cryostat, which comprises a microtome inside a freezer.
  • the surgical specimen is placed on a metal tissue disc which is then secured in a chuck and frozen rapidly to about -20°C to about -30°C.
  • the specimen is embedded in a gel like medium consisting of, for example, poly ethylene glycol and polyvinyl alcohol.
  • the frozen tissue is cut frozen with the microtome portion of the cryostat, and the section is optionally picked up on a glass slide and stained.
  • the tumor specimen may be a biopsy sample, such as cultured cells. These cells may be processed using the usual cell culture techniques that are known in the art. These cells may be circulating tumor cells.
  • the tumor specimen may be a biopsy sample, such as a formalin-fixed paraffin-embedded (FFPE) tumor tissue specimen.
  • FFPE formalin-fixed paraffin-embedded
  • a biopsy specimen may be placed in a container with formalin (a mixture of water and formaldehyde) or some other fluid to preserve it.
  • formalin a mixture of water and formaldehyde
  • the tissue sample may be placed into a mold with hot paraffin wax. The wax cools to form a solid block that protects the tissue.
  • This paraffin wax block with the embedded tissue is placed on a microtome, which cuts very thin slices of the tissue.
  • the tumor specimen contains less than 100 mg of tissue, or in certain embodiments, contains about 50 mg of tissue or less.
  • the tumor specimen (or biopsy) may contain from about 20 mg to about 50 mgs of tissue, such as about 35 mg of tissue.
  • the tissue may be obtained, for example, as one or more (e.g., 1, 2, 3, 4, or 5) needle biopsies (e.g., using a 14-gauge needle or other suitable size).
  • the biopsy is a fine-needle aspiration in which a long, thin needle is inserted into a suspicious area and a syringe is used to draw out fluid and cells for analysis.
  • the biopsy is a core needle biopsy in which a large needle with a cutting tip is used during core needle biopsy to draw a column of tissue out of a suspicious area.
  • the biopsy is a vacuum-assisted biopsy in which a suction device increases the amount of fluid and cells that is extracted through the needle.
  • the biopsy is an image-guided biopsy in which a needle biopsy is combined with an imaging procedure, such as, for example, X ray, computerized tomography (CT), magnetic resonance imaging (MRI) or ultrasound.
  • an imaging procedure such as, for example, X ray, computerized tomography (CT), magnetic resonance imaging (MRI) or ultrasound.
  • CT computerized tomography
  • MRI magnetic resonance imaging
  • ultrasound ultrasound
  • the sample may be obtained via a device such as the MAMMOTOME® biopsy system, which is a laser guided, vacuum-assisted biopsy system for breast biopsy.
  • tissue sample e.g., a biopsy sample or surgical specimen
  • tissue sample e.g., a biopsy sample or surgical specimen
  • This mechanical fragmentation of the explant may take place in a medium substantially free of enzymes that are capable of digesting the explant. Some enzymatic digestion may take place in certain embodiments.
  • the tissue sample may be systematically minced using two sterile scalpels in a scissor-like motion, or mechanically equivalent manual or automated opposing incisor blades. This cross-cutting motion creates smooth cut edges on the resulting tissue multicellular particulates.
  • the tumor particulates each measure from about 0.25 to about 1.5 mm 3 , for example, about 1 mm 3 .
  • the particles may be plated in culture flasks.
  • the number of explants plated per flask may vary, for example, between about one and about 25, such as from about 5 to about 20 explants per flask.
  • the explants may be evenly distributed across the bottom surface of the flask, followed by initial inversion for about 10 to about 15 minutes.
  • the flask may then be placed in a non-inverted position in a 37°C CC incubator for about 5 to about 10 minutes. Flasks are checked regularly for growth and contamination. Over a period of a few weeks a cell monolayer will form.
  • tumor cells grow out from the multicellular explant prior to stromal cells.
  • a predetermined time e.g., at about 10 to about 50 percent confluency, or at about 15 to about 25 percent confluency
  • growth of the tumor cells (as opposed to stromal cells) into a monolayer is facilitated.
  • the tumor explant may be agitated to substantially release tumor cells from the tumor explant, and the released cells cultured to produce a cell culture monolayer. The use of this procedure to form a cell culture monolayer helps maximize the growth of representative tumor cells from the tissue sample.
  • tumor/malignancy markers can be used in the present invention to assess cancer risk in patient including, but not limited to, one or more of MYBL2, MKI67, MAD2L1, AURKA, BCL2, BUB1, BIRC5, ESR1, CENPN, CCNB1, ERBB2, MLF1IP, NUDT1, PLK1, RNASE4, GGH, RRM2, CKS2, MCM4, CDKN3, Cl6orf6l, DLG7, H2AFZ, PFKP, KPNA2, GAT A3, CENPF, KRT18, KRT5, CCNE2, MELK, CX3CR1, TRIP13, MCM6, CCND1, PDIA4, CENPA, UBE2S, NCF1, CDC25B, PGR, TGFB3, PSMD2, HMMR, XBP1, TROAP, KNTC2, PRAME, BTG2, KRT8, FOXM1, KYNU, NME1, MCM3, NUSAP1, PCTK1, IGFBP5, CDC2, ER
  • the tumor/malignancy markers that can be used in the present invention to assess cancer risk include, but are not limited to, one or more of ALK gene rearrangements, Alpha-fetoprotein (AFP), Beta-2-microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), BCR-ABL fusion gene, BRAF mutation V600E, CA15-3/CA27.29, CA19-9, CA- 125, Calcitonin, Carcinoembryonic antigen (CEA), CD20, Chromogranin A (CgA), Chromosomes 3, 7, 17, and 9p2l, Cytokeratin fragments 21-1, EGFR mutation analysis, Estrogen receptor (ER)/progesterone receptor (PR), Fibrin/fibrinogen, HE4, HER2/neu, Immunoglobulins, KIT, KRAS mutation analysis, Lactate dehydrogenase, Nuclear matrix protein 22, Prostate-specific antigen (PSA), Thyrog
  • AFP
  • the tumor/malignancy markers used in the present invention to assess cancer risk include, but are not limited to, one or more of SSX protein family members, e.g., SSX1, SSX4, SSX5 or fragments thereof, MSN-l, OXA, OXB, PTEN, LeY, CAGE, UPAR, Hepcidin, and KLK4.
  • SSX protein family members e.g., SSX1, SSX4, SSX5 or fragments thereof, MSN-l, OXA, OXB, PTEN, LeY, CAGE, UPAR, Hepcidin, and KLK4.
  • the tumor/malignancy markers used in the present invention to assess cancer risk include, but are not limited to, one or more of prostate-specific antigen (PSA), prostate-specific membrane antigen (PMSA), prostate secretory protein (PSP), prostate acid phosphatase (PAP), human glandular kallekrein 2 (HK-2), prostate stem cell antigen (PSCA) and PTI-l.
  • PSA prostate-specific antigen
  • PMSA prostate-specific membrane antigen
  • PSP prostate secretory protein
  • PAP prostate acid phosphatase
  • HK-2 human glandular kallekrein 2
  • PSCA prostate stem cell antigen
  • the tumor/malignancy markers used in the present invention to assess cancer risk include, but are not limited to, one or more of b- actin, g-actin, a-tubulin, cytokeratin, cytokeratin 8 (CK 8), cytoskeletal tropomyosin, F-actin capping protein, hsp 27, hsp 60, hsp 70, hsp 90, grp 78 (BIP), gp 96, gluthathione-S-transferase, gluthathione synthetase, superoxide dismutae, thioredoxin peroxidase, PA28a, ubiquitin thiolesterase, triosephosphate isomerase, aldose reductase, enoyl-CoA hydratase, a-enolase, annexin II, IV and V, stathmin, nicotinamide-N-methyltransferase, B23/nu
  • Representative cancers and/or tumors of the present invention may include, but are not limited to, a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian
  • Gene expression can be measured using, for example, low-to-mid-plex techniques, including but not limited to reporter gene assays, Northern blot, fluorescent in situ hybridization (FISH), and reverse transcription PCR (RT-PCR). Gene expression can also be measured using, for example, higher-plex techniques, including but not limited, serial analysis of gene expression (SAGE), DNA microarrays. Tiling array, RNA-Seq/whole transcriptome shotgun sequencing (WTSS), high-throughput sequencing, multiplex PCR, multiplex ligation-dependent probe amplification (MLPA), DNA sequencing by ligation, and Luminex/XMAP.
  • SAGE serial analysis of gene expression
  • WTSS RNA-Seq/whole transcriptome shotgun sequencing
  • MLPA multiplex ligation-dependent probe amplification
  • DNA sequencing by ligation and Luminex/XMAP.
  • RNA products of the biomarkers within a sample, including arrays, such as microarrays, RT-PCR (including quantitative PCR), nuclease protection assays and Northern blot analyses.
  • arrays such as microarrays, RT-PCR (including quantitative PCR), nuclease protection assays and Northern blot analyses.
  • the disclosure includes in certain approaches methods of inhibiting development of genetically altered variants from genetically altered cells in cancer cell populations, wherein the cells have previously acquired a high degree of genomic instability (such as, but not limited to, cells that express functional LINE1 DNA repeats).
  • the disclosure includes in certain aspects targeting genetically altered cells with RT inhibitors and/or vaccines or other immunomodulating agents that selectively target genetically altered cells.
  • genetically altered cells include cells that acquired at least one of: point mutations, insertions, deletions, and amplifications, due at least in part to the activity of RT.
  • Genetically altered cells can include cells that have acquired novel processed pseudogenes, and cells that, because of their genetic modification, become resistant to cancer treatment or acquire other properties which confer selective advantages to such cells.
  • Genetically altered cells include but are not necessarily limited to cells of melanoma, neuroblastoma or other cancers that acquired amplification of protooncogenes of, for example, MYC family genes, (i.e..
  • CMYC, NMYC), and breast, ovarian or prostate cancer that have acquired amplified HER2/NEU genes, and cells of prostate cancer that have acquired amplification or point mutations in an androgen receptor gene, and thereby have become castration resistant, and other examples of treatment resistance cancer cells.
  • Genetically altered cells include cells with chromosomal abnormalities that distinguish them from other cells in a population, including cells with chromotripsis, etc.
  • the disclosure pertains to cells that have developed resistance to one or more cancer treatments, which means the cells have the ability to continue proliferation in the presence of chemotherapeutic drug(s), targeted anticancer agent(s), ongoing immunotherapy or radiation treatment to which the cells have developed resistance.
  • the present invention contemplates genetically altered cells comprising alternative lengthening of telomeres (ALT) induced or mediated by L-l (LINE-l) retrotransposon encoded reverse transcriptase.
  • the genetically altered cells are tumorigenic ALT cells.
  • the genetic instability of the present invention does not include alternative lengthening of telomeres (ALT) induced or mediated by L-l (LINE-l) retrotransposon encoded reverse transcriptase.
  • the genetically altered cells contemplated by the present invention are not tumorigenic ALT cells.
  • any inhibitor of reverse transcriptase can be used in methods of this disclosure.
  • any pharmaceutical agent(s) that possess inhibitory activity against polymerase and/or endonuclease activities against reverse transcriptase encoded by LINE elements can be used.
  • the RT is encoded by ORF2 of LINE1, or is encoded by ORF2 of LINE2 elements that are present in mammalian genomes.
  • Suitable RT inhibitors include but are not limited to those RT inhibitors of HIV, which possess cross reactivity with RT encoded by ORF2 LINE1 (i.e.. stavudine).
  • Suitable agents also include inhibitors of integrase of HIV, and accordingly possess cross reactivity with endonuclease activity of RT encoded by ORF2 of LINE1.
  • the RT inhibitor can be a nucleoside analog or nucleotide analog RT inhibitor, or a non-nucleoside or non-nucleotide RT inhibitor.
  • an RT inhibitor can comprise a compound or complex of compounds having dual inhibitory effects against RT and integrase.
  • disclosure includes administering to an individual in need thereof one or more compounds that function as Nucleoside analog reverse-transcriptase inhibitors (NARTIs or NRTIs), or Nucleotide analog reverse- transcriptase inhibitors (NtARTIs or NtRTIs).
  • NARTIs or NRTIs Nucleoside analog reverse-transcriptase inhibitors
  • NtARTIs or NtRTIs Nucleotide analog reverse- transcriptase inhibitors
  • Non-limiting examples of such compounds include AZT (Zidovudine), Didanosine, Zalcitabine, Stavudine, Lamivudine, Abacavir, Emtricitabine, Entecavir, Tenofovir, Adefovir, zalcitabine (ddC), lamivudine (3TC), and emtricitabine (FTC), abacavir (ABC) and entecavir (ETV), didanosine (ddl), tenofovir (TDF), and adefovir (ADV).
  • AZT Zadovudine
  • Didanosine Zalcitabine
  • Stavudine Lamivudine
  • Abacavir Emtricitabine
  • Entecavir Tenofovir
  • Adefovir zalcitabine
  • ddC lamivudine
  • FTC emtricitabine
  • ABSC abacavir
  • ETV did
  • NRTIs Non-nucleoside reverse-transcriptase inhibitors
  • the disclosure includes administering Portmanteau inhibitor.
  • patients are administered an adjuvant, neoadjuvant, or combination therapy with the RT inhibitor.
  • the adjuvant, neoadjuvant, or combination therapy can include a chemotherapy regimen (including, for example, monotherapy and combination therapies).
  • the chemotherapy may be, for example but is not limited to, paclitaxel, doxorubicin, mithramycin, docetaxel, platinum-based chemotherapeutics (including, but not limited to, cisplatin and carboplatin), mitomycin, methotrexate, fluorouracil, 5-fluorouracil (5-FU), vinorelbine, topotecan, irinotecan, bleomycin, bleomycin hydrorxyurea, mitomycin, actinomycin, topoisomerase I and II inhibitors, anthracybnes, epirubicin, idarubicin, mitoxantrone, valrubicin, etoposide, teniposide, rubitecan, and derivatives thereof.
  • the chemotherapy may include a taxane and/or an antimetabolite and/or derivatives thereof.
  • a patient may receive a chemotherapy selected from an anthracycbn, taxol or taxoid, vinca alkaloid, alkylating agent, intercalating agent, kinase inhibitor, or nitrogen mustard.
  • a chemotherapy selected from an anthracycbn, taxol or taxoid, vinca alkaloid, alkylating agent, intercalating agent, kinase inhibitor, or nitrogen mustard.
  • Non-limiting illustrative agents include one or more of a topoisomerase inhibitor (I or II), apoptosis inducer, protease inhibitor, microtubule inhibitor, mitotic inhibitor, an antimetabolite, signal transduction inhibitor, estrogen receptor inhibitor, EGFR inhibitor, Her2 inhibitor, or an aromatase inhibitor.
  • a patient may receive a chemotherapeutic agent that includes one or more of daunorubicin, doxorubicin, epirubicin, idarubicin, adriamycin, vincristine, carmustine, cisplatin, 5-fluorouracil, tamoxifen, prodasone, sandostatine, mitomycin C, foscamet, paclitaxel, docetaxel, gemcitabine, fludarabine, carboplatin, leucovorin, tamoxifen, goserebn, ketoconazole, leuprobde flutamide, vinblastine, vindesine, vinorelbine, camptothecin, topotecan, irinotecan hydrochloride, etoposide, mitoxantrone, teniposide, amsacrine, merbarone, piroxantrone hydrochloride, methotrexate, 6-mercaptopur
  • a patient may receive one or more of anti-Her2/neu antibodies such as HERCEPTIN, an anti-EGFR antibody such as ERBITUX, a growth factor receptor antibody such as AVASTIN, a small molecule inhibitor such as TARCEVA, IRESSA, or sunitinib), or anti-CD20 such as RITUXAN.
  • anti-Her2/neu antibodies such as HERCEPTIN, an anti-EGFR antibody such as ERBITUX, a growth factor receptor antibody such as AVASTIN, a small molecule inhibitor such as TARCEVA, IRESSA, or sunitinib
  • a patient receives erlotinib, gefitinib, lapatinib, cetuximab, panitumumab, or imatinib.
  • the disclosure includes methods of identification of new inhibitors of the endonuclease activity of RT LINE1 comprising screening candidate agents for their ability to inhibit RT, and/or for their ability to block DNA a damage response, such as in experimentally generated cells in culture carrying any type of a reporter indicative of DNA damage response (e.g., p53-responsive reporters).
  • the disclosure relates to pharmacological and immunotherapeutic approaches for inhibition and/or prevention of pathological consequences stemming from accumulation in normal somatic cell populations of mammalian body cell variants capable of spontaneously generating genetic alterations independently of cell divisions.
  • pathological consequences can comprise cancer, other proliferation disorders (i.e., myelodysplastic syndrome, benign tumors) and immunomodulation (i.e.. induction of inflammation) that may contribute to aging and malignant transformation.
  • a frailty index can be performed according to known approaches.
  • frailty is a state of increased vulnerability to adverse outcomes.
  • An FI index of this disclosure can include measuring any suitable number of accumulated deficits, such as at between 5-30 deficits.
  • a frailty index as described in Searle, et al, (2008) DOI: 10.1186/1471-2318-8-24, can be used, the disclosure of which is incorporated herein by reference.
  • a frailty index is calculated as described in U.S. patent publication no. 20150285823, from which the description of frailty index, frailty index parameters, and frailty index determination is incorporated herein by reference.
  • Frailty index parameters can include but are not limited to of weight, grip strength, blood pressure, complete blood count, and cytokine analysis.
  • frailty comprises an accumulation of deficiencies in major physiological functions, reduction of regeneration capabilities, impaired wound healing and increased risk of age-related diseases.
  • frailty is associated with natural aging or accelerated aging. Frailty can be measured according to any number of indices or tests known to one of skill in the art.
  • the Physiological Frailty Index includes measurement of one or more parameters selected from grip strength, systolic blood pressure, diastolic blood pressure, blood flow volume, number of blood neutrophils, percentage of blood neutrophils, number of blood monocytes, percentage of blood monocytes, number of lymphocytes, number of red blood cells, hemoglobin levels, hematocrit levels, mean corpuscular volume, mean corpuscular hemoglobin levels, mean corpuscular hemoglobin concentration and keratinocyte-derived cytokine levels. Deviation from a reference standard in any one individual is known as a deficit, and the overall average PFI score of the individual is a ratio of deficits to the total number of parameters measured.
  • PFI Physiological Frailty Index
  • Frailty can manifest as vulnerability to stressors and a reduced capacity to withstand stress.
  • the disclosure of Buchner and Wagner 1992 Clin Geriatr Med. 1992 Feb;8(l): l-l7 is hereby incorporated by reference in its entirety.
  • Frailty can manifest as loss of complexity of homeostatic mechanisms (e.g., interconnectedness and/or feedback or feedforward).
  • the disclosure of Lipsitz 2002 J Gerontol A Biol Sci MedSci. 2002 Mar;57(3):Bl l5- 25. is hereby incorporated by reference in its entirety.
  • Frailty can also manifest as disuse and/or a decrease in energy flow through an organism, as described in Bortz 2002, J Gerontol A Biol Sci Med Sci.
  • Frailty can also manifest as homeostatic dysregulation, as described by Ferrucci 2005 J. Gerontol. A Biol. Sci. Med. Sci. 60, 56, which is hereby incorporated by reference in its entirety.
  • FI Frailty Index
  • provided herein includes methods for improving and/or treating or preventing frailty and/or reducing frailty index in a patient.
  • Frailty can be assessed in any of many methods known in the art. For example, frailty and methods to evaluate/index frailty are described in Hubbard, et al, Ageing, published electronically November, 2008 page 115-118; Cesari, et al, Age and Ageing, 43: 10-12, 2014; and Mohler et al, Experimental Gerontology, 54:6-13, 2014, all of which are hereby incorporated by reference.
  • a Frailty Index is calculated as described in U.S. Patent Application Publication No. 2015/0285823, which is incorporated herein by reference. For example, a description of the determination of the Frailty Index is provided.
  • the Frailty Index was developed to assess a fit to frail range for the organisms of the same chronological age to address the notion that chronological age does not always reflect biologic age.
  • sixteen-item parameters that include measurements of weight, grip strength, blood pressure, complete blood count, cytokine level analysis
  • a FI is provided as a useful tool for assessing a“fit” to“frail” range organisms of the same chronological age.
  • methods of the present invention reduce or prevent frailty in a subject as measured according to the Physiological Frailty Index (PFI), as described in Antoch et al. Aging. 2017; 9: 1-12 (hereby incorporated by reference in its entirety).
  • PFI Physiological Frailty Index
  • PFI can be determined for an individual subject with reference to a young reference subject.
  • various parameters are measured. These parameters include non-invasive measurements, including age, body weight, grip strength, and diastolic blood pressure.
  • Additional blood chemistry measurements may also be determined, including white blood cell count, neutrophil count, neutrophil percentage, lymphocyte percentage, monocyte percentage, eosinophil percentage, red blood cell count, hemoglobin levels, hematocrit levels, mean corpuscular volume, mean corpuscular hemoglobin levels, mean corpuscular hemoglobin concentration, platelet count, and mean platelet volume.
  • STDEV standard deviation
  • Values that are different for one STDEV are scored as 0.25 (minimal deficit). Values that differ from the corresponding values in the reference group by 2 STDEV are scored as 0.5 and those that differ by 3 STDEV are scored as 0.75. If the value is above 3 STDEV, it is scored as 1 (extreme deficit). The number of deficits the individual subject expressed is calculated as a ratio of the total number of parameters measured and is referred to as Physiological Frailty Index (PFI).
  • PFI Physiological Frailty Index
  • methods of the present invention reduce or improve and/or treat or prevent frailty in a subject, as measured by the PFI.
  • a subject’s PFI score is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
  • a subject’s PFI score is reduced by about 25%-75%, about 25%-50%, or about 50% to 75%.
  • a subject’s PFI score is reduced to no greater than 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1 or 0.5.
  • frailty as an accumulation of deficits can be measured by the Rockwood frailty index, as described in Rockwood et al, JGer ontol A Biol SciMed Sci. 2007 Jul;62(7):722-727, which is incorporated by reference in its entirety.
  • the present methods reduce or prevent frailty as assessed by the Rockwood frailty index.
  • the Fried frailty score comprises a Physical Frailty Phenotype (PFP), which measures various parameters, such as weight loss of more than 10 pounds; weakness as related to grip strength; self-reported exhaustion; 15 feet walking speed; and amount of physical activity in Reals per week.
  • PFP Physical Frailty Phenotype
  • the Fried frailty score incorporates scoring of 0 (not frail), 1-2 (intermediate frailty), and greater than or equal to 3 (frail).
  • methods of the present invention reduce or improve and/or treat or prevent frailty in a subject, as measured by a Fried frailty score.
  • methods of the present invention can reduce or improve and/or treat or prevent frailty resulting in a reduced Fried frailty score from 3 to 2, from 3 to 1, from 3 to 0, from 2 to 1, from 2 to 0 or from 1 to 0.
  • Frailty can also be measured by the FRAIL Scale, as described in Abellean Van Kan et al. , J Am Med Dir Assoc. 2008 Feb;9(2):7l-2. doi: l0. l0l6/j.jamda.2007. l l.005, which is incorporated by reference in its entirety.
  • the parameters measured in the FRAIL Scale include feelings of persistent fatigue; resistance (ability to climb a single flight of stairs); ambulation (ability to walk one block); more than five illnesses; and more than 5% loss of weight.
  • the FRAIL Scale incorporates scoring of 0 (not frail), 1-2 (intermediate frailty), and greater than or equal to 3 (frail).
  • methods of the present invention reduce or improve frailty in a subject, as measured by a FRAIL Scale score.
  • methods of the present invention can reduce or improve frailty resulting in a reduced FRAIL Scale score from 3 to 2, from 3 to 1 , from 3 to 0, from 2 to 1 , from 2 to 0 or from 1 to 0.
  • the methods as provided herein improve (or reduce) frailty index, or delay or slow a decline in frailty using at least one accepted measure of fraility. In some embodiments the methods as provided herein improve (or reduce) frailty index, or delay or slow a decline in frailty using at least one accepted measure of fraility selected from the Frailty Index (FI), the Physiological Frailty Index (PFI), Fried frailty score, Rockwood frailty index, FRAIL Scale and the modified frailty index.
  • FI Frailty Index
  • PFI Physiological Frailty Index
  • Fried frailty score Rockwood frailty index
  • FRAIL Scale the modified frailty index.
  • the frailty comprises low lean mass, weakness, exhaustion, low energy expenditure and/or slow walking speed.
  • the present methods reduce or prevent the onset or development of one or more of low lean mass, weakness, exhaustion, low energy expenditure and/or slow walking speed.
  • compositions of this disclosure can comprise parenteral, intraperitoneal, intrapulmonary, oral, mucosal and topical administrations.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, and subcutaneous administration.
  • the amount of the RT inhibitor and/or immunogenic agent (or polynucleotide encoding it) and any other active agent to be included in a composition and/or to be used in the method can be determined by those skilled in the art, given the benefit of the present disclosure.
  • an effective amount of a composition of the invention is administered.
  • An effective amount can be an amount that that alleviates disease symptoms associated with cancer, aging, and/or age-related diseases.
  • an effective amount is such that as a consequence of vaccination at least some cells that express RT and/or comprise genetic instability as described herein are eliminated from an individual.
  • Effective amounts of RT inhibitors are those that inhibit RT function to reduce or eliminate creation of genetic instability that is described herein.
  • a composition comprising a vaccine and/or an RT inhibitor is administered to an individual in need thereof. The individual can be diagnosed with, suspected of having, or be at risk for any cancer, and/or for any age-related condition.
  • the disclosure is for prophylaxis and/or therapy for age-related diseases such as Alzheimer’s disease, type II diabetes, macular degeneration, chronic inflammation-based pathologies (e.g., arthritis), and/or to prevent development of cancer types known to be associated with aging (e.g., prostate cancer, melanoma, lung cancer, colon cancer, etc.), and/or with the purpose of improving the outcome of cancer treatment by radiation or chemotherapy.
  • age-related diseases such as Alzheimer’s disease, type II diabetes, macular degeneration, chronic inflammation-based pathologies (e.g., arthritis), and/or to prevent development of cancer types known to be associated with aging (e.g., prostate cancer, melanoma, lung cancer, colon cancer, etc.), and/or with the purpose of improving the outcome of cancer treatment by radiation or chemotherapy.
  • the individual treated using an approach of this disclosure is selected based at least in part on having cells comprising genetic instability, wherein the genetic instability is as further described herein.
  • the individual does not have, and/or has not
  • the individual is not HIV+ and has not been diagnosed as HIV+. In embodiments, the individual is characterized as having low risk of contracting an HIV infection, or another retroviral infection. In embodiments the individual is selected for treatment according to the present invention based on a determination of a frailty index, and/or by determining one or more parameters described herein that indicate genomic instability. In embodiments, the individual has not received an RT inhibitor before a first treatment with an RT inhibitor as described herein.
  • compositions administered according to this disclosure can vary depending on pharmaceutical formulation methods, administration methods, the patient’s actual or calculated age, body weight, sex, overall health, type and stage of cancer if cancer is being treated, diet, administration time, administration route, and other factors that will be apparent to those skilled in the art.
  • Compositions can be administered once, or over a series of administrations, and may be administered chronically so as to maintain genomic stability, and/or to improve genomic stability.
  • the disclosure comprises administering RT inhibitor(s) over an extended period of time, and thus can include chronic administration of effective doses of pharmacological agents capable of suppressing the polymerase and/or endonuclease function of RT LINE1.
  • Immunotherapeutic methods as described above comprise promoting selective killing of cells that express functional LINE1, and thus include vaccination approaches.
  • Vaccines of this disclosure can comprise compositions that include one or both proteins encoded by LINE1 elements (ORF1 and ORF2), and can include immunogenic fragments thereof.
  • the proteins can be combined with any suitable immunoadjuvant(s), including but not limited to alum and/or agonists of innate immunity receptors, and may be provided in association with such compositions, including non-covalent and covalent associations.
  • stimulation of an immune response can be achieved using any suitable recombinant vector(s) encoding one or both proteins of LINE1, and/or immunogenic fragments thereof.
  • Suitable recombinant vectors include but are not limited to adenoviral, lentiviral and adenoassociated viral vectors. Such vectors can also express immunomodulators (i.e.. bacterial flagellin) as immunoadjuvants.
  • immunomodulators i.e.. bacterial flagellin
  • cells that are targeted and reduced or eliminated from somatic cell populations by a stimulated immune system according to this disclosure include cells that express LINE1, or cells that have acquired novel processed pseudogenes or have chromosomal abnormalities.
  • genomic instability has not previously been considered as a treatment target itself since it has been viewed as a consequence of deficiencies in major pathways (DNA repair, replication control, mechanism restricting proliferation of damaged cells, etc.) without“druggable” targets.
  • the present disclosure introduces an alternative paradigm based on the interpretation that the genomic instability of tumor cells is caused by an active process involving desilencing and reverse transcriptase-driven amplification of repetitive retroelements that comprise (normally in silent form) nearly half of the mammalian genome.
  • RT reverse transcriptase
  • RT UNE LINE family
  • the disclosure relates in part to the observation that transcriptional desilencing of retroelements frequently occurs in cancer and can be provoked by loss of p53, which normally acts as an epigenetic repressor of the“DNA repeatome”
  • tumors frequently acquire activation of endogenous RT LINE expression that is not observed in normal cells besides early embryogenesis and neonatal brain.
  • a“genomic instability generator” RT as well as RNA templates for amplification
  • insertions integration of new copies of repeats and new processed pseudogenes
  • amplifications/deletions provoked by homologous recombination between newly integrated copies of repeats
  • point mutations via endonuclease activity of the integrase of RT LINE . Since all of these events are considered to be driven by a single enzyme - RT LINE - that is not expressed in normal cells, the disclosure relates to suppression of genomic instability in somatic cell populations by either inhibiting RT LINE activity with drugs or stimulating the immune system to eradicate cells with activated functional LINE elements.
  • the disclosure includes vaccines against antigens of LINEl-expressing cells which are tested for prevention of engraftment, and treatment of transplanted tumors expressing mouse LINE1.
  • Successful vaccines can demonstrate cancer preventive efficacy in four models of cancer-prone mice genetically predisposed to breast (MMTV-neu), prostate (PTEN hemizygous) or multiple cancers (p53 deficient, p53 hemizygous+irradiation).
  • the disclosure comprises targeting cells that comprise/express DNA retroelements.
  • These elements namely short and long interspersed nuclear elements (SINEs and LINEs), together comprise nearly 40% of most mammalian genomes. More than 65 million years ago, all major classes of SINEs and LINEs massively invaded the genomes of predecessors of mammals via a mechanism resembling naturally occurring PCR driven by reverse transcriptase (RT).
  • RT reverse transcriptase
  • the source of RT enzyme is provided by LINE1 elements ancient primitive RNA viruses that have only two open reading frames (ORFs), one of which, ORF2, encodes RT.
  • p53 cooperates with DNA methylation and a suicidal interferon response to maintain epigenetic silencing of repeats and noncoding RNAs. Proc Natl Acad Sci U SA 110, E89-98 (2013)). Nevertheless, all these safeguards cannot completely prevent activity of retroelements as evident from the analysis of tumors that frequently acquire expression of RT LINE by a few remaining functional elements among the -100,000 copies of LINEs present in the human or mouse genome. Tumors also commonly activate transcription of virus-like RNAs, thus providing RNA templates for RT UNE to initiate the process of amplification of retroelements. Consistently, appearance of newly integrated copies of both SINEs and LINEs has been demonstrated in many tumors and it has been shown that scale of RT activation correlates with stages of tumor progression.
  • retroelement activity is a major source of genomic instability leading to cancer development and progression an is part of an active ongoing process which, unlike other mechanisms involved in genomic instability (e.g., DNA replication inaccuracy, DNA repair inefficiency, etc.), is potentially druggable.
  • amplification of retroelements is believed to be completely dependent on the only known endogenous source of RT encoded by a few highly homologous copies of LINEls.
  • the present disclosure demonstrates that small molecule inhibitors of endogenous RT LINE create a decrease in the frequency of gene amplification in tumor cells in vitro and a delay in spontaneous tumor development in p53- deficient mice.
  • this disclosure indicates that RT activity can substantially affect genomic stability and its inhibition can reduce cancer development and progression.
  • this disclosure presents a paradigm-shifting strategy to cancer prevention, as well as addressing aging and age-related diseases, in which the plasticity cancer and other cells is approached as a druggable trait, and may provide for significant improvement of the efficacy of current treatments by suppressing development of drug-resistant tumor cell variants, as well as preventing and/or inhibiting spontaneous aging and age-related diseases.
  • genomic instability - the major property of cancer that makes it undefeatable - is a trait amenable to pharmacological and immunotherapeutic (vaccines) targeting.
  • the present disclosure relates to the concept of not targeting tumor viability (and other cells involved in aging and age-related disorders), but rather their “creativity” - genetic and phenotypic plasticity.
  • the disclosure defines a new role for RT inhibitors in oncology and aging as drugs for prevention of cancer development and progression, including acquisition of treatment-resistance, and related approaches and outcomes in the area of aging and age-related conditions. Further, in addition to its conceptual novelty, the present disclosure includes a number of important technical innovations.
  • the disclosure includes a retroposition-dependent suicidal element suitable for both in vitro use and in transgenic mice. Also included is a series of new rationally designed recombinant proteins for effective anti-LINE vaccination.
  • Vectors, genetically modified cell lines and transgenic mice resulting from this disclosure i.e., cell lines and mice with active RTs encoded by different constructs, molecular vaccines, etc.
  • LINE RT-driven amplification of retroelements is a major driver of genomic instability that contributes to both cancer origin and progression (acquisition of new properties such as drug resistance, metastatic capacity, etc.).
  • the disclosure also includes recognition of the influence of genomic instability on aging and age-related disorders.
  • RT-dependent genomic instability likely involves three types of events: (1) insertional mutagenesis caused by integration of new copies of LINEs and their RT-driven SINEs into functional areas of the genome, (2) deletions and amplifications provoked by homologous recombination between newly integrated repeats, and (3) point mutations triggered by the activity of LINE1 RT-associated endonuclease.
  • the current disclosure includes evaluating the effect of enforced expression of RT LINE in different contexts on genomic instability of somatic cells and to determine the contribution of each type of genome-destabilizing event in the overall mutagenic effect of RT LINE and to address specific mechanistic questions regarding the role of RT origin, contribution of ORF1, etc.
  • the disclosure accordingly includes generating vaccines and determining their (i) immunotherapeutic efficacy in experimental models of tumors with active LINEls, and (ii) immunoprophylactic efficacy in several models of cancer-prone mice.
  • the disclosure also relates to use of RT-inhibitors and such vaccines in methods of inhibiting aging and/or age-related disorders.
  • the disclosure includes use of assays to quantitatively assess the frequency of (i) retrotransposition, (ii) spontaneous gene amplification, and (iii) various gene-inactivating mutations (point mutations, deletions, etc.).
  • the first assay will utilize a recombinant vector named RTAD (for RetroTransposition Activity Detector) similar to the ORFEUS, a vector previously constructed to detect cells with ongoing retrotransposition.
  • RTAD is made from a mouse functional LINE element with ORF2 replaced by a reporter cassette consisting of puromycin-resistance and herpesvirus TK (ganciclovir sensitivity) genes under the ubiquitin promoter cloned in the orientation opposite to LINE1 transcription.
  • the promoter is inactivated by an intron.
  • puro and HSV TK expression is only possible if the intron is spliced out and a functional cDNA copy is synthesized from the spliced RNA. Since that latter step requires reverse transcription, RTAD serves as a detector of RT activity. Puromycin resistance will provide an indication of the frequency of cells in a population with functional RT. Ganciclovir selection can then be used to eradicate such cells and assess the impact of RT activity on genomic instability.
  • Gene amplification can be assessed by measuring the frequency of occurrence of cell variants resistant to N-phosphonoacetyl-L-aspartate (PALA), a small molecule inhibitor of aspartate transcarbamylase of CAD protein essential for UMP biosynthesis.
  • PALA N-phosphonoacetyl-L-aspartate
  • gene amplification frequency can be as high as 10-5 in tumor cells.
  • the frequency of gene-inactivating mutations can be determined using another series of detector cell lines transduced with lentiviral construct PURTK expressing the above-described puro-HSV TK reporter cassette conferring puromycin resistance and ganciclovir sensitivity.
  • the frequency of occurrence of ganciclovir-resistant clones in a cell population is a quantitative indicator of HSV TK gene inactivation by mutations. Therefore, these cells can be used to assess the effect of different factors on spontaneous mutagenicity.
  • RT LINE1 activity detector tools Two types can be generated from the same sets of mouse tumor-derived cell lines. These tools can be used to assess the dependence of three types of genomic instability - insertional mutagenesis, gene amplification and point mutations - on the activity of RT from different sources: (i) full-length LINE (natural and strong promoter-enforced ORF1 and ORF2 coexpression), (ii) RT line1 alone (to determine the impact of ORF1), and (iii) RT of different origin (MoMuLV RT - alone or within LINE1 in the place of ORF2). The nature of expected mutational events in selected cells can be analyzed as described herein.
  • a set of“modifier” constructs can be used to determine the ability of RT expression to provoke spontaneous transformation in vitro.
  • Two model systems that have been successfully used in our previous work will be used, both involving conversion of mouse Balb/c 3T3-derived non- transformed fibroblasts (clones (12)1 and (10)1 into transformed anchorage-independent tumorigenic cells.
  • (10)1 cells are p53-deficient and prone to transformation with activated mutant Ras while (12)1 cells are p53-wt and require p53 inactivation for Ras-mediated transformation. Both can be transfected with all modifier constructs either alone (line (12)1) or in combination with V-Ha-Ras under the SV40 promoter (line (12)1). Frequencies of cells in transduced populations capable of anchorage-independent growth due to acquisition of mutations either activating dominant oncogenes or suppressing p53 (in the case of (10)1 and (12)1, respectively) can be measured.
  • RTAD will provide a powerful new genetic tool not only for monitoring the activity of retroelements, but also for controlling genomic stability in cell populations by eradicating cells with activated repeats.
  • the disclosure includes creating transgenic mice carrying RTAD in their germline for use as a model for in vivo assessment of the feasibility of eradicating RT LINE1 -expressing cells for cancer prevention, as well as aging and age-related disorders.
  • the disclosure encompasses approaches to induction of adaptive immunity against cells with active retroelements and to assess their efficacy for cancer prophylaxis and therapy.
  • the disclosure encompasses all vaccines that can function in methods of the disclosure.
  • the disclosure provides two different types of vaccines: (i) protein-based (split vaccine) and (ii) gene-based adenoviral vaccine, and combinations of such approaches for use in, for example, prime-boost regimens.
  • the disclosure includes structural optimization of LINEl’s ORF1 and ORF2 which can include removal of immunosuppressive T-reg epitopes and adjusting codon contents to improve antigens’ expression levels.
  • ORF1 and ORF2 Genetic sequences encoding secreted forms of ORF1 and ORF2 can be created by, for example, addition of leader peptides according to established approaches.
  • various combinations of molecular adjuvants representing agonists of pattern-recognition receptors can be included in split vaccine. Immunogenicity of developed gene-based vaccines can be increased by addition into the adenoviral vector additional ORF encoding entolimod - a derivative of Salmonella flagellin, an agonist of TLR5 and powerful immunoadjuvant.
  • Vaccine formulations can be tested for their ability to activate cellular components of immunity, such as by FACS-based detection of lymphocyte activation markers (CD 107, CD69, CD25, CD44), analysis of proliferative response and cytokine production (IFN-g, IL-2, TNF) of antigen-specific CD4+ and CD8+ T lymphocytes upon their stimulation with ORF1 and ORF2 antigens.
  • Anti-tumor efficacy of both gene-based and split vaccine formulations can be tested using any suitable approaches, including but not limited to in vivo tumor models that include genetically modified tumor cells described above, and expressing LINEl’s ORF1/ORF2 antigens along with RTAD and grown as s.c.
  • Efficacy of anti-LINEl vaccines for prophylaxis of spontaneous cancers can be tested in established genetic models of mice predisposed to breast (MMTV-neu), prostate (PTEN hemizygous) or multiple cancers (p53 deficient, p53 hemizygous with total body irradiation). Animals can be immunized with vaccine preparations around 12 weeks of age and monitored for cancer development along with placebo control groups. Animals will be reserved for intermediate analysis at times of detectable hyperplastic lesions in target organs. Implementation of these approaches is expected to demonstrate substantial prophylactic and treatment effects of anti-LINEl vaccines (extension of tumor-free life, tumor growth suppression or regression). Expression of LINE1 antigens can be compared in tumors growing in vaccinated and control animals.
  • Figure 1 provides, without intending to be constrained by any particular theory, a schematic description of a new paradigm that sets forth new methods of anticancer and antiaging treatments that is based on pharmacological and immunotherapeutic targeting of cells expressing reverse transcriptase encoded by LINE1 family of elements (LINE1 RT).
  • Figure 1 schematically depicts cancer origin and progression, as well as aging, in mammals as a result of spontaneous epigenetic derepression of endogenous retrotransposons and their expansion in the genome via mechanism driven by LINE1 RT.
  • LINE1 RT enables synthesis and integration of cDNA copies of a variety of cellular RNA including RNA of LINE1 and SINE retrotransposons.
  • LINE1 RT-induced mutagenesis leads to accumulation of genetically and phenotypically altered cancer-initiating cells. If this process proceeds in a cancer cell population, it accelerates tumor progression involving acquisition of new malignant and treatment resistant properties. In populations of normal somatic cells, it leads to accumulation of cells with proinflammatory properties (e.g., senescent cells) and drives age-related increase in spontaneous sterile systemic inflammation.
  • proinflammatory properties e.g., senescent cells
  • LINE1 RT inhibitors including small molecules capable of inhibition of polymerase and/or endonuclease activity of this multifunctional enzyme, when applied to healthy organism, are expected to suppress aging and cancer development and can be used for cancer and aging prophylaxis. When applied to cancer-bearing organism, such inhibitors suppress cancer ability to accumulate additional mutations thereby slowing down cancer progression.
  • the RT inhibitors are expected to be suitable for use combination with any cancer treatment to make the treatment more effective via suppression of acquisition of treatment resistance.
  • FIG. 2 This Example is summarized in Figure 2, which provides evidence showing the ability of a representative anti-HIV drug, HIV RT inhibitor stavudine, to inhibit retrotranspositions of LINE1 elements within non-toxic range of concentrations.
  • Retrotransposition assay was performed according to Moran et al. Cell. 1996 87(5):9l7-27 (scheme shown to the left). HeLa cells were transfected with the reporter plasmid; appearance of neo-resistant colonies is indicative of retrotransposition events. Stavudine is effective at concentrations above 5mM. Lower panel demonstrates lack of effect of the indicated concentrations of stavudine on clonogenicity of HeLa cells. It is accordingly expected that other RT inhibitors will have similar utility.
  • Sensitivity of HCT116 cells to RT inhibitors alone were determined using quantitation of adherent cells following 72 hours of incubation in the presence of a range of concentrations of RT inhibitors (curves in the graph with the scale of cell viability shown on the right).
  • mice were kept on non-acidified drinking water, with or without 1.5 mg/ml of stavudine, either continuously or during indicated times. As evident from the graphs, stavudine alone had no detectable antitumor effect. Treatment with combination of cyclophosphamide and topotecan prolonged tumor-free life of mice for, on average, 4 weeks followed by tumor regrowth and animal termination due to critical tumor size. Continuous treatment with stavudine significantly delayed tumor relapses extending average tumor-free survival nearly two-fold and significantly increased the proportion of disease free animals with no relapses detected within >100 days of observation. These results support prospective use of RT inhibitors as suppressors of tumor progression in combination with standard anticancer regimens.
  • mice develop mammary gland tumors spontaneously starting from 7-th month of age. Tumors form in mammary pads and can be detected by palpation or visually during regular monitoring of animals. Twelve mice were selected as a control group and 15 mice as STV treatment group. All animals were bom during a period of 2 weeks. The data of tumor appearance are presented in Figure 6.
  • mice in STV group were on chronic drug administration for more than a year, they did not develop any signs of toxicity or any other phenotypic or behavioral differences besides slight differences in the dynamic of mouse weight: mice in STV group gained weight slower than in control group resulting in -10% differences in average weight of animals after 40 weeks on STV.
  • mice that received 17-DMAG in combination with STV had a statistically significant delay in tumor relapse as compared to mice that received only 17-DMAG.
  • a patient that has never been diagnosed with cancer and has also never been diagnosed with HIV or any retroviral infection visits a licensed physician and the physician diagnosis the patient as having a genetic trait that increases susceptibility to cancer.
  • the doctor prescribes an RT inhibitor to the patient and monitors the patient. The patient remains cancer free.

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Abstract

La présente invention concerne au moins en partie la prévention du cancer ou la prévention d'une rechute du cancer chez un patient. Dans certains modes de réalisation, les procédés de l'invention concernent la prévention d'un cancer primaire et/ou la prévention d'une rechute du cancer chez un patient par administration d'un inhibiteur de la transcriptase inverse (RT) au patient.
PCT/US2019/038205 2018-06-20 2019-06-20 Prévention du cancer primaire et résistant au traitement par des inhibiteurs de la transcriptase inverse endogène Ceased WO2019246376A1 (fr)

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US20220260465A1 (en) * 2021-02-17 2022-08-18 Sysmex Corporation Sample scraping method and sample scraping device
WO2022271880A1 (fr) * 2021-06-22 2022-12-29 Rome Therapeutics, Inc. Procédés de traitement d'états médicaux et d'inhibition de la transcriptase inverse line1 à l'aide d'un acide adéninyl-propyloxy phosphonique substitué ou d'un composé apparenté
WO2023178128A1 (fr) * 2022-03-15 2023-09-21 Rome Therapeutics, Inc. Méthodes de traitement d'états médicaux à l'aide d'islatravir ou d'un composé associé
US12187758B2 (en) 2022-03-15 2025-01-07 Rome Therapeutics, Inc. Compounds and methods for treating disease

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220260465A1 (en) * 2021-02-17 2022-08-18 Sysmex Corporation Sample scraping method and sample scraping device
US11927516B2 (en) * 2021-02-17 2024-03-12 Sysmex Corporation Sample scraping method and sample scraping device
WO2022271880A1 (fr) * 2021-06-22 2022-12-29 Rome Therapeutics, Inc. Procédés de traitement d'états médicaux et d'inhibition de la transcriptase inverse line1 à l'aide d'un acide adéninyl-propyloxy phosphonique substitué ou d'un composé apparenté
WO2023178128A1 (fr) * 2022-03-15 2023-09-21 Rome Therapeutics, Inc. Méthodes de traitement d'états médicaux à l'aide d'islatravir ou d'un composé associé
US12187758B2 (en) 2022-03-15 2025-01-07 Rome Therapeutics, Inc. Compounds and methods for treating disease

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