WO2024064192A1 - Treatment of cellular proliferative disease via dsif complex modulation, and compositions for practicing the same - Google Patents

Treatment of cellular proliferative disease via dsif complex modulation, and compositions for practicing the same Download PDF

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WO2024064192A1
WO2024064192A1 PCT/US2023/033228 US2023033228W WO2024064192A1 WO 2024064192 A1 WO2024064192 A1 WO 2024064192A1 US 2023033228 W US2023033228 W US 2023033228W WO 2024064192 A1 WO2024064192 A1 WO 2024064192A1
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protein
spt4
subject
cells
terra
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French (fr)
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Stanley N. Cohen
Weijing Xu
Yanan Feng
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The Board Of Trustees Of The Leland Stanford Junior University
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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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • Spt4 Transcription elongation factor Spt4 was first identified in Saccharomyces cerevisiae [Winston, F., 1984], and is highly conserved among eukaryotes [Guo, M. 2008; Hartzong, G.A., 1996; Wenzel, B.M., 2010].
  • the DRB sensitivity-inducing factor (DSIF) complex formed by Spt4 and Spt5 has been implicated in multiple steps of RNAPII transcription, including promoter proximal pausing, elongation, and RNA processing/termination [Hartzog, 2013].
  • Spt4 enhances RNA polymerase II processivity by reducing dissociation of polymerase from highly structured regions of DNA template, most frequently in the repeat region.
  • Telomeres are nucleoprotein complexes that protect the physical ends of linear chromosomes from degradation and unscheduled repair activities [Lingner, J., 1995; Sfeir, A. 2012], Telomeres were considered transcriptionally silent regions of mammalian chromosomes until Azzalin et al.
  • TERRA Telomeric Repeat-containing RNA
  • TERRA molecules are comprised of subtelomere and telomere-derived UUAGGG repeats and vary in length from 100 bases to 9 kb in mammals [Azzalin, C.M., 2007].
  • TERRA has been proved to play critical roles in telomere biology, including regulation of telomere length, telomerase activity and heterochromatin formation at chromosome ends [Cusanelli, E., 2013].
  • TERRA transcription initiates from subtelomeric promoters located on at least two- thirds of chromosome ends [Nergadze, S., 2009; Deng Z, 2012; Porro A, 2014], Those subtelomeric promoters comprise CpG dinucleotide-rich DNA islands, characterized by 61 - 29-37 repeats upstream of TERRA Transcription Start Site (TSS) and at ⁇ 1 kb from the telomeric repeat region [Brown WRA, 1990; Nergadze, 2009].
  • TSS TERRA Transcription Start Site
  • TERRA harbors long UUAGGG repeats (corresponding to telomeric TTAGGG repeats) transcribed from highly structured telomere regions, it is speculated that the elongation activity of RNA polymerase II could regulate TERRA transcription.
  • a transcription elongation factor has been identified as a new regulator of TERRA in yeast [Rodrigues J, 2018].
  • Paf1 and Ctr9 two components of yeast transcription elongation complex PAF1 , help to maintain low TERRA levels, and further analysis of Paf1 and Ctr9 double mutants showed that high TERRA levels are correlated with poor fitness through mechanisms that are independent of previously identified regulators of TERRA such as Sir4, Rati , and Trf4.
  • Methods of treating a subject for a cellular proliferative disease are provided. Aspects of the methods include: administering to the subject an agent that modulates DSIF complex activity, e.g., activity of a DSIF complex made up of a SPT4 and SPT5 protein, such as a DSIF complex made up of Supt4h and Supt5h, in a manner sufficient to treat the subject for the cellular proliferative disease.
  • an agent that modulates DSIF complex activity e.g., activity of a DSIF complex made up of a SPT4 and SPT5 protein, such as a DSIF complex made up of Supt4h and Supt5h, in a manner sufficient to treat the subject for the cellular proliferative disease.
  • compositions for practicing the methods are also provided.
  • U2OS cells were treated with 20nM, 6.6nM, 2.2nM supt4 siRNA and control siRNA for 5 days and 10 days respectively, RNA was extracted and analyzed.
  • A TERRA repeat abundance were analyzed by dot blot using 3 2P-y[ATP]-labeled probe generated by endlabeling of (AATCCC)4 oligo. The levels of input RNA were normalized to 18S RNA.
  • B Quantification analysis of the dot-blotting results in (A).
  • C Quantitative real-time analysis of abundance of subtelomere sequences from individual chromosome specific TERRA in U2OS cells treated with 20nM Supt4 siRNA for 10 days.
  • U2OS cells were transfected with Ienti-supt4 shRNA, and control empty virus with puromycin resistance (puro Ctrl) respectively for 20 days, genomic DNA was extracted and analyzed by C-circle formation (A) and Quantitative real-time PCR analysis of telomere repeat abundance (B). The levels of Supt4 transcript and protein in U2OS cells treated with Ienti-supt4 and puro control virus were detected in (C) and (D), GAPDH was used as internal control.
  • Figure 4 The size and abundance of telomeres in U2OS cells treated with lenti- supt4 determined by genomic TRF assay.
  • U2OS cells were transfected with Ienti-supt4 shRNA, and control empty virus with puromycin resistance (puro Ctrl) respectively for 20 days, genomic DNA was extracted and analyzed by Southern-TRF with 32 P-Y[ATP]-labeled telomere G probe.
  • B Quantification of the signal in high MW region and low MW region was performed in Typhoon software.
  • FIG. 5 Supt4 siRNA knock-down increases cell apoptosis in ALT + U2OS cells, but not in ALT’ Hela cells.
  • U2OS cells (A) and Hela cells (B) were treated with 10nM supt4 siRNA for 10 days before cells were collected for FACS analysis.
  • Alexa Fluor 488 Annexin V/Dead cell apoptosis kit was used to analyze the status of each cell. Cells were stained with two dyes: Annexin V stains apoptotic cells by its ability to bind to phosphatidylserine, and PI stains dead cells. Recombinant Annexin V is conjugated to Alexa Fluor 488, which is a perfect spectral match to fluorescein (FITC).
  • FITC fluorescein
  • Q1 PI-, FITC+: apoptotic cells
  • Q2 PI+, FITC+: late apoptotic cells
  • Q3 PI+, FITC-: necrotic cells
  • Q4 PI-, FITC-: viable cells
  • A SAOS-2, MG-63, and U2OS cells were treated with 10 nM supt4 siRNA and control siRNA for 10, RNA was extracted and TERRA repeat abundance were analyzed by dot blot using 32 P-Y[ATP]-labeled probe generated by end-labeling of (AATCCC) 4 oligo. The levels of input RNA were normalized to 18S RNA.
  • B Supt4 transcript abundance in the various knock-down strains.
  • FIG. 7 Supt4 siRNA knock-down decreases telomerase activity in ALT- Hela cells. Quantitative TRAPeze-RT analysis of telomerase activity using CHAPS extracted cellular protein extract. Ct. values of real-time reaction were converted into TPG units using a standard curve generated by dilution series of telomerase substrate oligonucleotide TSR8 with eight telomeric repeats. A TSV template was used as internal control of PCR amplification. Heated samples served as the negative control for telomerase (labeled as with heat).
  • A Relative telomerase activity of normally growing Hela (ALT-), U2OS (ALT+), and BJ (fibroblast) cells.
  • B Relative telomerase activity of Hela cells treated with supt4 siRNA for 5 days.
  • FIG. 8 Supt4 RNA and protein abundance in U2OS cells treated with supt4 siRNA.
  • U2OS cells were treated with 20 nM, 6.6 nM, 2.2 nM supt4 siRNA and control siRNA for 5 days and 10 days respectively.
  • A RNA was extracted and analyzed for the relative levels of Supt4 transcript. GAPDH was used as internal control.
  • B Protein samples from U2OS cells treated with 6.6 nM supt4 siRNA for 5 and 10 days were analyzed by Western-blotting for Supt4 protein abundance.
  • FIG. 9 72 hours Supt4 siRNA knock-down has no effect on ALT in U2OS cells.
  • U2OS cells were treated with 10nM supt4 siRNA, control siRNA, and lipofectamine RNAimax (lipo) respectively for 72 hours, genomic DNA was extracted and analyzed.
  • A Genomic TRF assay to determine the size and abundance of telomeres.
  • B Quantitative real-time PCR analysis of telomere repeat abundance.
  • C Detection of C-circles by CC assay using serial dilutions (1/3) of reaction products.
  • D The relative levels of Supt4 transcript in U2OS cells treated with supt4 siRNA and control siRNA for 72 hours. GAPDH was used as internal control.
  • FIG. 11 Effect of supt4 siRNA KN on C circle formation in U2OS and SAOS- 2.
  • SAOS-2 and U2OS cells were treated with 10 nM supt4 siRNA and control siRNA for 5 days and 10 days respectively.
  • DNA was extracted and analyzed for C-circles by CO assay using serial dilutions (1/3) of reaction products.
  • HD143 treatment affects the proliferation of U2OS and MG-63.
  • the cells were treated with different doses of HD143 and its derivative CPD25 for 72 hours.
  • Titer-glow assay was used to measure the viable cells after the compound treatment.
  • Hela cells were treated with 10nM, 2 nM, 0.4 nM supt4 siRNA and control siRNA respectively for 5 days and 10 days in 10cm culture plate. The total cell number was counted by vi-cell.
  • Methods of treating a subject for a cellular proliferative disease are provided. Aspects of the methods include: administering to the subject an agent that modulates DSIF complex activity, e.g., activity of a DSIF complex made up of a SPT4 and SPT5 protein, such as a DSIF complex made up of Supt4h and Supt5h, in a manner sufficient to treat the subject for the cellular proliferative disease.
  • an agent that modulates DSIF complex activity e.g., activity of a DSIF complex made up of a SPT4 and SPT5 protein, such as a DSIF complex made up of Supt4h and Supt5h, in a manner sufficient to treat the subject for the cellular proliferative disease.
  • compositions for practicing the methods are also provided.
  • Cellular proliferative diseases are conditions characterized by abnormal cellular proliferation, such as cancers. Such disease conditions include cancer and neoplastic diseases and other diseases characterized by the presence of unwanted cellular proliferation, e.g., hyperplasias, and the like.
  • the cellular proliferative disease is a cancer, and in some instances is an ALT+ cancer.
  • the methods include administering to the subject an agent that modulates DSIF complex activity in a manner sufficient to treat the subject for the cellular proliferative disease.
  • the target DSIF complex in such embodiments may vary, where in some embodiments the target DSIF complex comprises an SPT4 protein and an SPT5 protein, such as a DSIF complex comprising Supt4h and Spt5h.
  • Active agents of interest include, but are not limited to, small molecules, nucleic acids (e.g., DNA, RNA), proteins (e.g., antibodies, enzymes, nucleases, proteases, etc.), and the like, or any combination thereof.
  • the agent modulates the activity of the protein following expression, such that the agent is one that changes the activity of the protein encoded by the target gene following expression of the protein from the target gene. In these instances, the agent is one that may act directly with protein encoded by the target gene.
  • the agent may be one that selectively reduces the deleterious activity, e.g., transcription of extremely long repeat regions in non-genic DNA, of the encoded protein, but retains or enhances, at least to a detectable level, the beneficial activity of the encoded protein.
  • such agents are not inhibitors of the protein, but instead selectively reduce the deleterious activity of the protein via another mechanism, e.g., by reducing the amount of the protein in the cell that is available for interaction with other proteins, by reducing production of the protein, , by preventing assembly of a protein complex, etc.
  • the agent modulates expression of the RNA and/or protein from the gene, such that it changes the expression of the RNA or protein from the target gene in some manner.
  • the agent may change expression of the RNA or protein in a number of different ways.
  • DSIF complex modulating agents of interest include selective SPT4 modulatory agents.
  • Selective SPT4 modulatory agents are agents that selectively change the SPT4 activity in a cell, e.g., decrease SPT4 activity in a cell.
  • the target SPT4 activity that is modulated, e.g., decreased, by the active agent is a transcription activity, and specifically an activity that facilitates RNA polymerase II processivity through target repeat domains, e.g., long TTAGGG repeat domains.
  • the target SPT4 activity that is modulated by such agents is an activity arising from an SPT4 protein.
  • SPT4 protein is used herein to collectively refer to not only yeast Spt4 proteins, but also mammalian homologs thereof, e.g., human SUPT4H; murine Supt4h, etc.
  • SPT4 proteins of interest whose activity may be modulated by the selective SPT4 modulatory agents include, but are not limited to, S. cerevisiae Spt4; human SUPT4H and murine Supt4h.
  • the agent employed in methods of the invention is an SPT4 modulatory agent
  • the modulatory agent that is employed may be any agent that, upon introduction into a cell, changes the SPT4 activity of the cell, and specifically reduces the SPT4-mediated extended target repeat transcription activity in the subject.
  • the SPT4 modulatory agent may modulate activity in a number of different ways, e.g., by reducing expression of an SPT4 protein, by inhibiting binding of an SPT4 protein to another protein, e.g., a protein interacting with SPT4 (e.g., an SPT5 protein, such as Supt5h (Spt5 or SUPT5H)), by preventing SPT4 from forming a DSIF complex with SPT5, etc.
  • SPT4 modulatory agent may modulate activity in a number of different ways, e.g., by reducing expression of an SPT4 protein, by inhibiting binding of an SPT4 protein to another protein, e.g., a protein interacting with SPT4 (e
  • the agent is one that reduces, including inhibits, expression of a functional SPT4 protein.
  • Inhibition of SPT4 protein expression may be accomplished using any convenient means, including use of an agent that inhibits SPT4 protein expression, such as, but not limited to, antisense agents, RNAi agents, agents that interfere with transcription factor binding to a promoter sequence of the SPT4 gene, or inactivation of the SPT4 gene, e.g., through recombinant techniques, etc.
  • antisense molecules can be used to down-regulate expression of an SPT4 gene in the cell.
  • the anti-sense reagent may be antisense oligodeoxynucleotides (ODN), particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA.
  • ODN antisense oligodeoxynucleotides
  • the antisense sequence is complementary to the mRNA of the targeted protein, allowing the antisense sequence to hybridize to the mRNA and form a duplex that inhibits expression of the targeted protein.
  • Antisense molecules inhibit gene expression through various mechanisms, e.g., by reducing the amount of mRNA available for translation, through cleavage of the RNA:DNA heteroduplex by activation of RNAse H, or steric hindrance.
  • One or a combination of antisense molecules may be administered, where a combination may include multiple different sequences.
  • Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the transcriptional initiation is oriented such that an antisense strand is produced as an RNA molecule.
  • the antisense molecule is a synthetic oligonucleotide.
  • Antisense oligonucleotides will generally be at least about 7, usually at least about 12, more usually at least about 20 nucleotides in length, and not more than about 500, usually not more than about 50, more usually not more than about 35 nucleotides in length, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like. It has been found that short oligonucleotides, of from 7 to 8 bases in length, can be strong and selective inhibitors of gene expression (see Wagner et al. (1996), Nature Biotechnol. 14:840-844).
  • Targets for antisense molecules may be a specific region or regions of the endogenous sense strand mRNA sequence that are chosen to be complemented by the antisense sequence. Selection of a specific sequence for the oligonucleotide may use an empirical method, where several candidate sequences are assayed for inhibition of expression of the target gene in an in vitro or animal model. A combination of sequences may also be used, where several regions of the mRNA sequence are selected for antisense complementation.
  • Antisense oligonucleotides may be chemically synthesized by methods known in the art (see Wagner et al. (1993), supra, and Milligan et al., supra.) Oligonucleotides may be chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases.
  • phosphorothioates Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates.
  • Achiral phosphate derivatives include 3'-0'-5'-S-phosphorothioate, 3'-S-5'-0-phosphorothioate, 3'-CH 2 -5'-O- phosphonate and 3'-NH-5'-0-phosphoroamidate.
  • Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity.
  • the a-anomer of deoxyribose may be used, where the base is inverted with respect to the natural (3-anomer.
  • the 2'-OH of the ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl sugars, which provides resistance to degradation without comprising affinity. Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl- 2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. 5- propynyl-2'- deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.
  • catalytic nucleic acid compounds e.g. ribozymes, anti-sense conjugates, etc. may be used to inhibit gene expression.
  • Ribozymes may be synthesized in vitro and administered to the patient, or may be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell (for example, see International patent application WO 9523225, and Beigelman et al. (1995), Nucl. Acids Res. 23:4434-42). Examples of oligonucleotides with catalytic activity are described in WO 9506764.
  • Conjugates of anti-sense ODN with a metal complex, e.g. terpyridylCu(ll), capable of mediating mRNA hydrolysis are described in Bashkin et al. (1995), Appl. Biochem. Biotech not. 54:43-56.
  • RNAi agents e.g., double-strand RNA (Sharp (1999) Genes and Development 13: 139-141).
  • RNAi such as double-stranded RNA interference (dsRNAi) or small interfering RNA (siRNA)
  • dsRNAi double-stranded RNA interference
  • siRNA small interfering RNA
  • RNAi agents may be dsRNA or a transcriptional template of the interfering ribonucleic acid which can be used to produce dsRNA in a cell.
  • the transcriptional template may be a DNA that encodes the interfering ribonucleic acid.
  • Additional RNAi mediators include shRNA, microRNAs (miRNA), and artificial miRNAs. Methods and procedures associated with RNAi are also described in WO 03/010180 and WO 01/68836, all of which are incorporated herein by reference.
  • dsRNA can be prepared according to any of a number of methods that are known in the art, including in vitro and in vivo methods, as well as by synthetic chemistry approaches. Examples of such methods include, but are not limited to, the methods described by Sadher et al. (Biochem. Int.
  • Single-stranded RNA can also be produced using a combination of enzymatic and organic synthesis or by total organic synthesis.
  • the use of synthetic chemical methods enables one to introduce desired modified nucleotides or nucleotide analogs into the dsRNA.
  • dsRNA can also be prepared in vivo according to a number of established methods (see, e.g., Sambrook, et al.
  • RNA can be directly introduced intracellularly.
  • RNA delivery by microinjection see, e.g., Zernicka-Goetz, et al. (1997) Development 124:1133-1137; and Wianny, et al. (1998) Chromosoma 107: 430-439).
  • Other options for cellular delivery include permeabilizing the cell membrane and electroporation in the presence of the dsRNA, liposome-mediated transfection, or transfection using chemicals such as calcium phosphate.
  • a number of established gene therapy techniques can also be utilized to introduce the dsRNA into a cell. By introducing a viral construct within a viral particle, for instance, one can achieve efficient introduction of an expression construct into the cell and transcription of the RNA encoded by the construct.
  • the SPT4 gene is inactivated so that it no longer expresses a functional protein.
  • inactivated is meant that the gene, e.g., coding sequence and/or regulatory elements thereof, is genetically modified so that it no longer expresses a functional SPT4 protein, e.g., at least with respect to SPT4 transcription activity through a region of extended target repeats.
  • expression of the SPT4 protein may be reduced or inhibited.
  • a non-functional (e.g., mutated) SPT4 protein may be expressed.
  • the alteration or mutation may take a number of different forms, e.g., through deletion of one or more nucleotide residues, through exchange of one or more nucleotide residues, and the like.
  • Various methods of gene editing may be employed, including e.g., those methods capable of introducing a single nucleotide substitution, those methods capable of introducing a site-directed deletion and those methods capable of introducing site-directed insertion.
  • Useful site-directed gene editing methods described in more detail below, include methods that employ a nuclease to cleave one or both strands of a target nucleic acid molecule.
  • the cleaved target nucleic acid may be subsequently repaired, e.g., through homology directed repair (HDR), to introduce the edit at a desired location.
  • HDR homology directed repair
  • Methods for generating targeted gene modifications through homologous recombination are known in the art, including those described in: U.S. Patent Nos. 6,074,853; 5,998,209; 5,998,144; 5,948,653; 5,925,544; 5,830,698; 5,780,296; 5,776,744; 5,721 ,367; 5,614,396; 5,612,205; the disclosures of which are herein incorporated by reference.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • TALEN Transcription Activator-Like Effector Nuclease
  • ZFN Zinc Finger Nuclease
  • Methods of site-directed introduction of a desired edit will vary and may include introducing a site directed cleavage event, e.g., through the use of a site-directed nuclease (e.g., a CRISPR/Cas9 nuclease, a TALEN nuclease, a ZFN, and the like) followed by a specific repair event at the site cleaved by the site-directed nuclease.
  • a site-directed nuclease e.g., a CRISPR/Cas9 nuclease, a TALEN nuclease, a ZFN, and the like
  • Such methods of specific repair may include, e.g., homologous recombination, including homology directed repair (HDR).
  • HDR homology directed repair
  • Also of interest in certain embodiments are dominant negative mutants of SPT4 proteins, where expression of such mutants in the cell result in a modulation, e.g., decrease, in SPT4 mediated transcription of extended target repeats in a cell.
  • Dominant negative mutants of SPT4 are mutant proteins that exhibit dominant negative SPT4 activity.
  • the term "dominant-negative SPT4 activity" or “dominant negative activity” refers to the inhibition, negation, or diminution of certain particular activities of SPT4, and specifically to SPT4 mediated transcription of extended target repeats. Dominant negative mutations are readily generated for corresponding proteins.
  • mutant polypeptide may interact with wild-type polypeptides (made from the other allele) and form a non-functional multimer. In certain embodiments, the mutant polypeptide will be overproduced. Point mutations are made that have such an effect.
  • fusion of different polypeptides of various lengths to the terminus of a protein, or deletion of specific domains can yield dominant negative mutants.
  • RNA capable of encoding gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, M. J. ed., IRL Press, Oxford.
  • the agent is an agent that modulates, e.g., inhibits, SPT4 activity by binding to SPT4 and/or inhibiting binding of SPT4 to a second protein, e.g., an SPT5 protein, such as Spt5 or SUPT5H.
  • SPT4 protein is used herein to collectively refer to not only yeast Spt4 proteins, but also mammalian homologs thereof, e.g., human SUPT4H; murine Supt4h, etc.
  • SPT4 proteins of interest whose activity may be modulated by the selective SPT4 modulatory compounds include, but are not limited to, S. cerevisiae Spt4; human SUPT4H and murine Supt4h.
  • SPT4 modulatory agents are compounds that change the SPT4 activity in a cell, e.g., decrease SPT4 activity in a cell.
  • the compound may be a selective SPT4 modulatory agent.
  • the target SPT4 activity that is modulated, e.g., decreased, by the active compound is a transcription activity, and specifically an activity that facilitates RNA polymerase II processivity through a target repeat.
  • the target SPT4 activity that is modulated by such compounds is an activity arising from an SPT4 protein.
  • the compound that is employed may, upon introduction into a cell, change the SPT4 functionality in the cell, and at least differentially reduce the SPT4 mediated extended target repeat transcription activity in the subject.
  • the SPT4 modulatory agent may modulate functionality in a variety of ways, e.g., by inhibiting binding of an SPT4 protein to another protein, e.g., a protein interacting with SPT4 (e.g., an SPT5 protein, such as Spt5 or SUPT5H), etc.
  • the subject compound diminishes interaction of the SPT4 protein and a second protein.
  • the second protein is a SPT5 protein.
  • SPT5 protein is used herein to collectively refer to not only yeast Spt5 proteins, but also mammalian homologs thereof, e.g., human SUPT5H; murine Supt5h, etc.
  • the subject compound diminishes interaction between Supt4h and Supt5h.
  • Human Supt4h may form a DSIF complex with Supt5h, as may its yeast ortholog, to regulate transcription elongation (Guo et al., "Core structure of the yeast spt4-spt5 complex: a conserved module for regulation of transcription elongation," Structure (2008) 16: 1649-1658; Hatzog et al., " Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae," Genes Dev.
  • the compound diminishes interaction between RNA polymerase II and Supt5h or the DSIF complex.
  • a subject compound may interfere with binding of Supt 5h to RNA polymerase II, and its effects on the interaction between Supt4h and Supt5h may be indirect.
  • the second protein is a SPT5 protein (e.g., as described herein).
  • the extent of binding of the SPT4 protein to the second protein is reduced by 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or by 100% , e.g., as compared to a suitable control, e.g., a cell not contacted with the compound of interest. Any convenient methods may be utilized to determine extent of binding of the SPT4 protein to the second protein.
  • the compound diminishes interaction between Supt4h and Supt5h and disrupts the subsequent formation of the DSIF complex.
  • the compound may specifically bind to the SPT4 protein and disrupt the interaction of the SPT4 protein with the SPT5 protein. In some instances, the compound specifically binds to the SPT5 protein and disrupts the interaction between the SPT4 and SPT5 protein. In some cases, by disrupting the interaction between the SPT4 and STP5 protein, the formation of the DSIF complex is also disrupted.
  • an effective amount of a compound is an interaction diminishing amount, i.e. , an amount of the compound that inhibits the formation of a SPT4 complex (e.g., a SPT4/SPT5 (DSIF) complex) by 20% or more, such as 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more, as compared to SPT4 complex formation in the absence of the compound.
  • a SPT4 complex e.g., a SPT4/SPT5 (DSIF) complex
  • Any convenient methods of assaying inhibition of complex formation or competitive inhibition may be utilized, such as those methods described by Cheng et al.
  • the subject compound modulates expression of extended repeat regions or domains that are not in genes (i.e. , that are in non-genic DNA), such as telomeric regions, e.g., telomeric regions that contain extended repeats, e.g., telomeric TTAGGG repeat containing regions.
  • telomeric regions e.g., telomeric regions that contain extended repeats, e.g., telomeric TTAGGG repeat containing regions.
  • the method reduces expression of such repeat regions, such as reducing expression of TERRA from telomeric repeat regions.
  • the method reduces transcription elongation, but not transcription initiation.
  • the method reduces the number of repeats transcribed in a repeat containing region such that the generated transcripts are relatively shorter and have fewer repeats than the transcripts from a control, e.g., the method reduces the number of repeats present in each TERRA transcript. In some instances, the method reduces the abundance of high molecular weight transcripts, e.g., high molecular weight TERRA transcripts.
  • any convenient assays may be used to determine a reduction in expression or a reduction in the number of repeats in the transcripts in a cell using the subject compounds relative to a control, e.g., a cell not contacted with the compound of interest, where the magnitude of expression reduction may be 10% or more, such as 20% or more, 30% or more, 50% or more, 100% or more, such as by 2-fold or more, by 5- fold or more, by 10-fold or more, by 20-fold or more, by 50-fold or more, by 100-fold or more, or even more.
  • the magnitude of any difference in expression resulting from administration of the compound may vary, where in some instances the magnitude of reduction of expression relative to corresponding expression in a control is 2-fold or more, by 5- fold or more, by 10- fold or more, by 20-fold or more, by 50-fold or more, by 100-fold or more, or even more.
  • the methods may reduce the deleterious impact of expression of a non-genic nucleotide repeat domain, such as telomeric regions that contain extended repeats, e.g., telomeric TTAGGG repeat containing regions.
  • the subject compound reduces the ability of a cancer cell to use alternative lengthening of telomeres (ALT) pathways and break- induced replication (BIR) mechanisms to support telomere elongation and cell immortality.
  • ALT telomeres
  • BIR break- induced replication
  • the subject compound reduces cell proliferation and growth in cancer cells.
  • the subject compound suppresses or inhibits telomerase activity.
  • any convenient assays may be used to determine the effect of using the subject compounds relative to a control, e.g., a cell not contacted with the compound of interest, where the magnitude of reduction may be 10% or more, such as 20% or more, 30% or more, 50% or more, 100% or more, such as by 2-fold or more, by 5- fold or more, by 10-fold or more, by 20-fold or more, by 50-fold or more, by 100-fold or more, or even more.
  • the magnitude of any differences resulting from administration of the compound may vary, where in some instances the magnitude of reduction relative to the corresponding effect in a control is 2-fold or more, by 5- fold or more, by 10-fold or more, by 20-fold or more, by 50-fold or more, by 100-fold or more, or even more.
  • the subject compound increases apoptosis of cancer cells, e.g., ALT+ cancer cells.
  • Any convenient assays may be used to determine the effect of using the subject compounds relative to a control, e.g., a cell not contacted with the compound of interest, where the magnitude of increase may be 10% or more, such as 20% or more, 30% or more, 50% or more, 100% or more, such as by 2-fold or more, by 5- fold or more, by 10-fold or more, by 20-fold or more, by 50-fold or more, by 100-fold or more, or even more.
  • the magnitude of any differences resulting from administration of the compound may vary, where in some instances the magnitude of increase relative to the corresponding effect in a control is 2-fold or more, by 5- fold or more, by 10-fold or more, by 20-fold or more, by 50-fold or more, by 100-fold or more, or even more.
  • an effective amount of an agent e.g., such as described above, e.g., an SPT4 modulatory agent
  • the effective amount of the compound is provided in the cell by contacting the cell with the compound.
  • Contact of the cell with the modulatory agent may occur using any convenient protocol. The protocol may provide for in vitro or in vivo contact of the modulatory agent with the target cell, depending on the location of the target cell. In some instances, the cell is in vitro. In certain instances, the cell is in vivo. Contact may or may not include entry of the compound into the cell.
  • the modulatory agent may be introduced directly into the cell under cell culture conditions permissive of viability of the target cell.
  • the choice of method is generally dependent on the type of cell being contacted and the nature of the compound, and the circumstances under which the transformation is taking place (e.g., in vitro, ex vivo, or in vivo).
  • the modulatory agent may be administered to the organism or subject in a manner such that the compound is able to contact the target cell(s), e.g., via an in vivo or ex vivo protocol.
  • in vivo it is meant in the target construct is administered to a living body of an animal.
  • ex vivo it is meant that cells or organs are modified outside of the body. Such cells or organs are in some cases returned to a living body.
  • the method is an in vivo method that includes: administering to a subject in need thereof an effective amount of an agent, e.g., as described above.
  • treating or “treatment” as used herein means the treating or treatment of a disease or medical condition in a patient, such as a mammal (such as a human) that includes: (a) preventing the disease or medical condition from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as, eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient.
  • the disease or medical condition to be treated is a cellular proliferative disease.
  • Cellular proliferative diseases are conditions characterized by abnormal cellular proliferation, such as cancers. Such disease conditions include cancer and neoplastic diseases and other diseases characterized by the presence of unwanted cellular proliferation, e.g., hyperplasias, and the like.
  • the cellular proliferative disease to be treated is cancer.
  • cancer cells To escape replicative senescence, cancer cells must overcome telomere attrition during DNA replication. Most cancer cells rely on telomerase to extend and maintain telomeres, but a significant fraction use the telomerase-independent pathway of alternative lengthening of telomeres (ALT) to overcome telomere shortening.
  • the cancer to be treated is a telomerase positive (TEL+) cancer. In some cases, the cancer to be treated is an alternative lengthening of telomeres positive (ALT+) cancer.
  • the cancer to be treated is an ALT+ cancer.
  • ALT+ cancers are characterized by the use of a homologous recombination-based ALT pathway to extend and maintain telomeres and acquire replicative immortality.
  • telomeres are elongated by break-induced replication (BIR), a repair process initiated by one-ended double-stranded breaks (DSBs) at collapsed replication forks and extended by conservative DNA replication.
  • BIR break-induced replication
  • DSBs double-stranded breaks
  • ALT is prevalent in cancers from the mesenchymal origin and usually associates with poor clinical outcome.
  • ALT activity in cells and ALT+ cancers may be identified by the detection of one or more biomarkers, including, but not limited to, an absence of telomerase or telomerase activity, the presence of telomeric variant repeats (e.g., TAAGGG, TTAGAG, and TTTGGG); telomeres of substantial length (e.g., >50 kb); telomeres of heterogeneous length (e.g., ⁇ 8 kb and >50 kb); elevated levels of telomere sister chromatid exchange (tSCE); extra-chromosomal telomeric repeats (ECTRs), particularly C-circles; ALT-associated acute promyelocytic leukemia (PML) bodies (APBs); and telomere dysfunction-induced foci (TIFs).
  • telomeric variant repeats e.g., TAAGGG, TTAGAG, and TTTGGG
  • telomeres of substantial length e.g.,
  • ALT Positivity in Human Cancers: Prevalence and Clinical Insights Cancers (Basel) (2021) 13(10): 2384).
  • cancers that may be ALT+ include, but are not limited to, osteosarcomas, breast cancers, gliomas, choroid plexus carcinomas, medullablastomas, neuroblastomas, pancreatic neuroendocrine tumors (PanNETs), angiosarcomas, leiomyosarcomas, lipsarcomas, and undifferentiated pleomorphic sarcomas (see Zhang et al. (2021 ), supra).
  • the terms “host”, “subject”, “individual” and “patient” are used interchangeably and refer to any mammal in need of such treatment according to the disclosed methods.
  • Such mammals include, e.g., humans, ovines, bovines, equines, porcines, canines, felines, non-human primate, mice, and rats.
  • the subject is a non-human mammal.
  • the subject is a farm animal.
  • the subject is a pet.
  • the subject is mammalian. In certain instances, the subject is human.
  • Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees, and monkeys).
  • domestic pets e.g., dogs and cats
  • livestock e.g., cows, pigs, goats, horses, and the like
  • rodents e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease
  • non-human primates e.g., chimpanzees, and monkeys.
  • the amount of compound administered can be determined using any convenient methods to be an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle.
  • the specifications for the unit dosage forms of the present disclosure will depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • an effective amount of a subject compound is an amount that ranges from about 50 ng/ml to about 50 pg/ml (e.g., from about 50 ng/ml to about 40 pg/ml, from about 30 ng/ml to about 20 pg/ml, from about 50 ng/ml to about 10 pg/ml, from about 50 ng/ml to about 1 pg/ml, from about 50 ng/ml to about 800 ng/ml, from about 50 ng/ml to about 700 ng/ml, from about 50 ng/ml to about 600 ng/ml, from about 50 ng/ml to about 500 ng/ml, from about 50 ng/ml to about 400 ng/ml, from about 60 ng/ml to about 400 ng/ml, from about 70 ng/ml to about 300 ng/ml, from about 60 ng/ml to about 100 ng/ml, from about 65 ng/ml,
  • an effective amount of a subject compound is an amount that ranges from about 10 pg to about 100 mg, e.g., from about 10 pg to about 50 pg, from about 50 pg to about 150 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 500 pg to about 750 pg, from about 750 pg to about 1 ng, from about 1 ng to about 10 ng, from about 10 ng to about 50 ng, from about 50 ng to about 150 ng, from about 150 ng to about 250 ng, from about 250 ng to about 500 ng, from about 500 ng to about 750 ng, from about 750 ng to about 1 pg, from about 1 pg to about 10 pg, from about 10 pg to about 50 pg, from about 50 pg to about 150 pg, from about 150 pg to about 250 pg, from about 250 pg to about to about 500
  • a single dose of the subject compound is administered.
  • multiple doses of the subject compound are administered.
  • the DSIF complex modulating compound is administered twice daily (qid), daily (qd), every other day (qod), every third day, three times per week (tiw), or twice per week (biw) over a period of time.
  • a compound is administered qid, qd, qod, tiw, or biw over a period of from one day to about 2 years or more.
  • a compound is administered at any of the aforementioned frequencies for one week, two weeks, one month, two months, six months, one year, or two years, or more, depending on various factors.
  • a biological sample obtained from an individual who has been treated with a subject method can be assayed for the presence and/or level of cells that are cancer cells, e.g., ALT+ cancer cells.
  • Assessment of the effectiveness of the methods of treatment on the subject can include assessment of the subject before, during and/or after treatment, using any convenient methods.
  • aspects of the subject methods further include a step of assessing the therapeutic response of the subject to the treatment.
  • the method includes assessing the condition of the subject, including diagnosing or assessing one or more symptoms of the subject which are associated with the disease or condition of interest being treated (e.g., as described herein).
  • the method includes obtaining a biological sample from the subject and assaying the sample, e.g., for the presence of a target gene or gene product or for the presence of cells that are associated with the disease or condition of interest (e.g., as described herein).
  • the sample can be a cellular sample. In some cases, the sample is a biopsy.
  • the assessment step(s) of the subject method can be performed at one or more times before, during and/or after administration of the subject compounds, using any convenient methods.
  • the assessment step includes measuring the RNA or protein expression levels of SPT4. In some cases, the assessment step includes directly measuring the activity of SPT4, e.g., assaying for the association of SPT4 with SPT5 to form a DSIF complex or for the association of the DSIF complex with RNA pol II. In some embodiments, the assessment step includes indirectly measuring the activity of SPT4, e.g., assaying for the presence or abundance of the DSIF complex, assaying for TERRA expression, assaying for the presence of ALT+ biomarkers, assaying for telomere length, or assaying for telomerase activity as described herein. In certain cases, the assessment step includes identification of cells including cancer cells.
  • the cancer cells are ALT+ cancer cells. In other embodiments, the cancer cells are TEL+ cells. In some cases, identification of cells includes determining whether the cells are apoptotic or viable or assaying for cell growth or proliferation. In certain instances, assessing the subject includes diagnosing whether the subject has a disease or condition of interest. In some instances, assessing the subject includes determining whether the subject has a symptom associated with the disease or condition of interest.
  • the method delays occurrence of a symptom associated with the disease. In certain instances, the method reduces the magnitude of a symptom associated with the disease.
  • surrogate marker is employed in its conventional sense to refer to a measure of the effects of specific disease treatment or predict outcomes in a clinical trial.
  • Surrogate markers can be defined as a laboratory measurement or a physical sign that is used in therapeutic trials as a substitute for a clinically meaningful endpoint.
  • Reliable surrogates, rigorously validated in phase III clinical trials, can forecast the long term effect of the therapy based on how the patient feels, functions, or survives (Katz, “Biomarkers and Surrogate Markers: an FDA Perspective,” NeuroRx (2004) 1 : 189-95).
  • markers may also be used to compare drug efficacy between trials and may even become the basis for which new drugs gain regulatory approval for marketing (Twaddell, “Surrogate outcome markers in research and clinical practice,” Australian Prescriber (2009) 32: 47-50). Because their use can reduce the size, duration, and cost of large studies or clinical trials, these markers are especially valuable if the predicted drug effect prevents death or promotes other critically important outcomes. For some progressive diseases, surrogate markers may be able to determine the disease stage (Weston, “The use of surrogate end points in cardiovascular disease and diabetes,” The British Journal of Cardiology (2008) 15: S6-S7). Depending on the specific disease condition, surrogate markers may vary widely. Embodiments of the present disclosure therefore include administering a compound, e.g., as described herein, to modulate, e.g., improve, one or more surrogate markers of the disease condition.
  • a compound e.g., as described herein
  • the compound e.g., as described herein
  • the subject compounds can be incorporated into a variety of formulations, e.g., pharmaceutically acceptable vehicles, for therapeutic administration.
  • the subject methods result in reduction in the deleterious activity of an extended target repeat in a target cell or cells, where the target cell(s) may be in vitro or in vivo.
  • the subject methods result in reduction in toxicity of a target gene, e.g., via a reduction in aggregation of a protein encoded thereby, in a target cell(s) .
  • the methods result in enhancement in function of a protein encoded by a target gene.
  • compositions that include an active agent (e.g., as described herein) (for example one or more of the subject compounds, either alone or in the presence of one or more additional active agents) present in a pharmaceutically acceptable vehicle.
  • an active agent e.g., as described herein
  • pharmaceutically acceptable vehicles may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans.
  • vehicle refers to a diluent, adjuvant, excipient, or carrier with which a compound of the present disclosure is formulated for administration to a mammal.
  • Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
  • the compounds and compositions of the present disclosure and pharmaceutically acceptable vehicles, excipients, or diluents may be sterile.
  • an aqueous medium is employed as a vehicle when the subject compound is administered intravenously, such as water, saline solutions, and aqueous dextrose and glycerol solutions.
  • compositions can take the form of capsules, tablets, pills, pellets, lozenges, powders, granules, syrups, elixirs, solutions, suspensions, emulsions, suppositories, or sustained-release formulations thereof, or any other form suitable for administration to a mammal.
  • the pharmaceutical compositions are formulated for administration in accordance with routine procedures as a pharmaceutical composition adapted for oral or intravenous administration to humans. Examples of suitable pharmaceutical vehicles and methods for formulation thereof are described in Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro ed., Mack Publishing Co.
  • Administration of the subject compounds may be systemic or local. In certain embodiments administration to a mammal will result in systemic release of a compound of the present disclosure (for example, into the bloodstream).
  • Methods of administration may include enteral routes, such as oral, buccal, sublingual, and rectal; topical administration, such as transdermal and intradermal; and parenteral administration.
  • Suitable parenteral routes include injection via a hypodermic needle or catheter, for example, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intraarterial, intraventricular, intrathecal, and intracameral injection and non-injection routes, such as intravaginal rectal, or nasal administration.
  • the compounds and compositions of the present disclosure are administered subcutaneously.
  • the compounds and compositions of the present disclosure are administered orally.
  • the compounds can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • a subject compound may also be formulated for oral administration.
  • suitable excipients include pharmaceutical grades of carriers such as mannitol, lactose, glucose, sucrose, starch, cellulose, gelatin, magnesium stearate, sodium saccharine, and/or magnesium carbonate.
  • the composition may be prepared as a solution, suspension, emulsion, or syrup, being supplied either in solid or liquid form suitable for hydration in an aqueous carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, preferably water or normal saline.
  • compositions suitable for oral administration can include (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, or saline; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
  • Lozenge forms can include the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles including the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are described herein.
  • an inert base such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are described herein.
  • the subject formulations can be made into aerosol formulations to be administered via inhalation.
  • These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They may also be formulated as pharmaceuticals for non-pressured preparations such as for use in a nebulizer or an atomizer.
  • formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Formulations suitable for topical administration may be presented as creams, gels, pastes, or foams, containing, in addition to the active ingredient, such carriers as are appropriate.
  • the topical formulation contains one or more components selected from a structuring agent, a thickener or gelling agent, and an emollient or lubricant.
  • Frequently employed structuring agents include long chain alcohols, such as stearyl alcohol, and glyceryl ethers or esters and oligo(ethylene oxide) ethers or esters thereof.
  • Thickeners and gelling agents include, for example, polymers of acrylic or methacrylic acid and esters thereof, polyacrylamides, and naturally occurring thickeners such as agar, carrageenan, gelatin, and guar gum.
  • emollients include triglyceride esters, fatty acid esters and amides, waxes such as beeswax, spermaceti, or carnauba wax, phospholipids such as lecithin, and sterols and fatty acid esters thereof.
  • the topical formulations may further include other components, e.g., astringents, fragrances, pigments, skin penetration enhancing agents, sunscreens (e.g., sunblocking agents), etc.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet, or suppository, contains a predetermined amount of the composition containing one or more inhibitors.
  • unit dosage forms for injection or intravenous administration may include the inhibitor(s) in a composition as a solution in sterile water, normal saline, or another pharmaceutically acceptable carrier.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present disclosure calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle.
  • the specifications for the novel unit dosage forms of the present disclosure depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • the compounds may be administered in the form of a free base, their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
  • Dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Desired dosages for a given compound are readily determinable by a variety of means.
  • the dose administered to an animal, particularly a human, in the context of the present disclosure should be sufficient to effect a prophylactic or therapeutic response in the animal over a reasonable time frame, e.g., as described in greater detail herein. Dosage will depend on a variety of factors including the strength of the particular compound employed, the condition of the animal, and the body weight of the animal, as well as the severity of the illness and the stage of the disease.
  • the size of the dose will also be determined by the existence, nature, and extent of any adverse sideeffects that might accompany the administration of a particular compound. COMBINATION T HERAPY
  • the DSIF complex modulating agents may be used jointly with any agent useful in the treatment of a neoplastic condition, such as anti-cancer agents and anti-tumor agents.
  • Agents of interest which can be used jointly with the subject DSIF modulating compounds in such instances include, but are not limited to, Cancer chemotherapeutic agents, Agents that act to reduce cellular proliferation, Antimetabolite agents, Microtubule affecting agents, Hormone modulators and steroids, natural products, and biological response modifiers, e.g., as described in greater detail below.
  • Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinaceous) compounds that reduce proliferation of cancer cells and encompass cytotoxic agents and cytostatic agents.
  • Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds can also be used.
  • Suitable cancer chemotherapeutic agents include dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like).
  • MMAD Monomethyl auristatin D
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci.
  • duocarmycins and active analogs and derivatives thereof e.g., including the synthetic analogues, KW-2189 and CB 1 -TM1
  • benzodiazepines and active analogs and derivatives thereof e.g., pyrrolobenzodiazepine (PBD).
  • PBD pyrrolobenzodiazepine
  • Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (CytoxanTM), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.
  • alkylating agents such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclopho
  • Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatine, and gemcitabine.
  • CYTOSAR-U cytarabine
  • cytosine arabinoside including, but not limited to, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercap
  • Suitable natural products and their derivatives include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.; antibiotics, e.g.
  • anthracycline daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclic immunosuppressants, e.g.
  • cyclosporine FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like.
  • Other anti-proliferative cytotoxic agents are navelbene, CPT-11 , anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
  • Microtubule affecting agents that have antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, epothilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.
  • Hormone modulators and steroids that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g.
  • adrenocorticosteroids e.g. prednisone, dexamethasone, etc.
  • estrogens and pregestins e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.
  • adrenocortical suppressants e.g.
  • estradiosteroids stimulate proliferation and differentiation. Therefore, compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids may inhibit T cell proliferation.
  • chemotherapeutic agents include metal complexes, e.g., cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g., hydroxyurea; and hydrazines, e.g., N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc.
  • metal complexes e.g., cisplatin (cis-DDP), carboplatin, etc.
  • ureas e.g., hydroxyurea
  • hydrazines e.g., N-methylhydrazine
  • epidophyllotoxin e.g., a topoisomerase inhibitor
  • procarbazine mitoxantrone
  • leucovorin tegafur
  • anti-proliferative agents of interest include immunosuppressants, e.g., mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4- morpholinyl)propoxy)quinazoline) ; etc.
  • immunosuppressants e.g., mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4- morpholinyl)propoxy)quinazoline) ; etc.
  • Taxanes are suitable for use.
  • “Taxanes” include paclitaxel, as well as any active taxane derivative or pro-drug.
  • “Paclitaxel” (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXOLTM, TAXOTERETM (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3’N- desbenzoyl-3’N-t-butoxycarbonyl analogs of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881 , WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S.
  • Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., TaxotereTM docetaxel, as noted above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose). Also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but not limited to, galactose and mannose derivatives described in International Patent Application No.
  • WO 99/18113 piperazino and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021 , WO 98/22451 , and U.S. Patent No. 5,869,680; 6- thio derivatives described in WO 98/28288; sulfenamide derivatives described in U.S. Patent No. 5,821 ,263; and taxol derivative described in U.S. Patent No. 5,415,869. It further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98/58927; WO 98/13059; and U.S. Patent No. 5,824,701.
  • Biological response modifiers suitable for use include, but are not limited to, (1 ) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine/threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-a; (7) IFN-y; (8) colony-stimulating factors; and (9) inhibitors of angiogenesis.
  • RTK tyrosine kinase
  • the agents of the invention are employed in combination with immunotherapy agents.
  • immunotherapy include anti-PD-1/PD-L1 immunotherapies, such as anti-PD-1/PD-L1 therapeutic antagonists, where such antagonists include but are not limited to e.g., OPDIVO® (nivolumab), KEYTRUDA® (pembrolizumab), TecentriqTM (atezolizumab), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS-936559 (MDX-1105), CA-170, BMS-202, BMS-8, BMS-37, BMS-242 and the like.
  • Nivolumab is a humanized lgG4 anti-PD-1 monoclonal antibody used to treat cancer.
  • Pembrolizumab formerly known as MK-3475, lambrolizumab, etc.
  • Atezolizumab TecentriqTM
  • Durvalumab Medlmmune is a therapeutic monoclonal antibody that targets PD-L1 .
  • Avelumab (also known as MSB0010718C; Merck KGaA, Darmstadt, Germany & Pfizer) is a fully human monoclonal PD-L1 antibody of isotype lgG1 .
  • BMS-936559 (also known as MDX-1105; Bristol-Myers Squibb) is a blocking antibody that has been shown to bind to PD-L1 and prevent its binding to PD-1 (see e.g., U.S. NIH Clinical Trial No. NCT00729664).
  • CA-170 (Curis, Inc.) is a small molecule PD-L1 antagonist.
  • BMS-202, BMS-8, BMS-37, BMS-242 are small molecule PD- 1/PD-L1 complex antagonists that bind PD-1 (see e.g., Kaz et al., (2016) Oncotarget 7(21); the disclosure of which is incorporated herein by reference in its entirety).
  • Anti-PD-L1 antagonists, including e.g., antibodies, useful in the methods described herein include but are not limited to e.g., those described in U.S. Patent Nos. 7,722,868; 7,794,710;
  • Anti-PD-1 antagonists including e.g., antibodies, useful in the methods described herein include but are not limited to e.g., those described in 6,808,710; 7,029,674; 7,101 ,550; 7,488,802; 7,521 ,051 ; 8,008,449; 8,088,905; 8,168,757; 8,460,886; 8,709,416; 8,951 ,518; 8,952,136; 8,993,731 ; 9,067,998; 9,084,776; 9,102,725; 9,102,727; 9,102,728; 9,109,034; 9,181,342; 9,205,148; 9,217,034; 9,220,776; 9,308,253; 9,358,289; 9,387,247 and 9,402,899; the disclosures of which are incorporated herein by reference in their entirety.
  • co-administration and “in combination with” include the administration of two or more therapeutic agents either simultaneously, concurrently, or sequentially within no specific time limits.
  • the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time.
  • the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms.
  • a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.
  • Conscomitant administration of a known therapeutic drug with a pharmaceutical composition of the present disclosure means administration of the compound and second agent at such time that both the known drug and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a subject compound. Routes of administration of the two agents may vary, where representative routes of administration are described in greater detail below. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compounds of the present disclosure.
  • the compounds are administered to the subject within twenty-four hours of each other, such as within 12 hours of each other, within 6 hours of each other, within 3 hours of each other, or within 1 hour of each other. In certain embodiments, the compounds are administered within 1 hour of each other. In certain embodiments, the compounds are administered substantially simultaneously. By administered substantially simultaneously is meant that the compounds are administered to the subject within about 10 minutes or less of each other, such as 5 minutes or less, or 1 minute or less of each other.
  • the subject methods and compound compositions find use in treatment of a subject for a disease condition, such as a cellular proliferative disease condition, e.g., a cancer, such as an ALT+ cancer.
  • a disease condition such as a cellular proliferative disease condition, e.g., a cancer, such as an ALT+ cancer.
  • treatment is meant at least an amelioration of one or more symptoms associated with the disease condition afflicting the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the pathological condition being treated, such as loss of cognitive function, etc.
  • treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition.
  • Treatment may also manifest in the form of a modulation of a surrogate marker of the disease condition, e.g., as described above.
  • hosts are treatable according to the subject methods.
  • Such hosts are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs and rats), and primates (e.g., humans, chimpanzees and monkeys).
  • the host is human.
  • the subject methods and compound compositions also find use in modulating transcription mediated by the DSIF complex comprising SPT4 and SPT5.
  • the subject methods and compound compositions also find use in modulating transcription elongation, reducing the transcription of RNA with extended repeat regions (e.g., TERRA, pathogenic RNA in HD or ALS, etc.) or the number of repeats transcribed, and research in transcription, ALT mechanisms, the role of TERRA, and telomere lengthening.
  • the subject methods and compound compositions find use in the characterization of ALT+ and ALT- cancers.
  • the subject methods and compound composition also find use in modulating telomerase dysfunction and treating associated cancers thereof.
  • kits and systems that find use in practicing embodiments of the methods, such as those described as described above.
  • system refers to a collection of two or more different active agents, present in a single or disparate composition, that are brought together for the purpose of practicing the subject methods.
  • kit refers to a packaged active agent or agents.
  • the subject system or kit includes a dose of a subject compound (e.g., as described herein) and a dose of a second active agent (e.g., as described herein) in amounts effective to treat a subject for a disease or condition associated with the deleterious activity of an extended nucleotide repeat containing target region.
  • Kits and systems for practicing the subject methods may include one or more pharmaceutical formulations.
  • kits may include a single pharmaceutical composition, present as one or more unit dosages, where the composition may include one or more nucleoside compounds (e.g., as described herein).
  • the kit may include two or more separate pharmaceutical compositions, each containing a different active agent, at least one of which is a nucleoside compound (e.g., as described herein).
  • kits and systems finding use in the subject methods, e.g., as described above.
  • Such kits and systems may include one or more components of the subject methods, e.g., nucleoside agents, cells, vectors encoding proteins of interest, enzyme substrates, dyes, buffers, etc.
  • the various kit components may be present in the containers, e.g., sterile containers, where the components may be present in the same or
  • subject kits may further include instructions for using the components of the kit, e.g., to practice the subject method.
  • the instructions are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e. , associated with the packaging or sub-packaging) etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, Hard Disk Drive (HDD), portable flash drive, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
  • Spt4/Spt5 is a highly conserved transcription elongation complex which regulates polymerase II processivity.
  • the human orthologue Supt4 is selectively required for the expression of long trinucleotide repeat containing transcripts in multiple neurodegenerative diseases models.
  • the function of Supt4 is further investigated for the transcription of the non-coding RNA TERRA, the (TTAGGG) 6 repeat sequence transcribed from the chromosome telomere region.
  • supt4 depletion decreases TERRA repeat abundance in U2OS, an osteosarcoma cell line which harbors extremely long TERRA due to the mechanism of Alternative Lengthening of Telomere, and consequently disrupts telomere recombination, shortens telomere and causes apoptosis.
  • the supt4 shRNA lenti-virus was added to the cell culture together with polybrene (8 pg/ml), and 1 pg/ml puromycin was used for selection.
  • Genomic DNA was extracted using DNeasy kit (Qiagen) according to manufacturer’s protocol.
  • 10pg of high molecular weight genomic DNA were digested with the Rsal and Hif I restriction enzymes (NEB biolabs). After electrophoresis in 0.6% agarose gel, DNA was denatured and transferred to nylon membrane (Amersham Hybond-N + , GE healthcare). The hybridization procedure was performed according to the protocol (Zhou S, 2011]. Briefly, The membranes were hybridized in 0.5M phosphate buffer with 7% SDS and 1 mM EDTA containing 3 2P-y[ATP]-labeled probe generated by endlabeling of (TTAGGG)4 oligo for 18 hours at 50°C . After hybridization, the membranes were washed in 4XSSC 0.1%SDS twice at room temperature, then in 2XSSC 0.1%SDS twice at 50°C. Radioactive signal were detected using a phosphoimager.
  • RNA from cancer cells was extracted using RNeasy kit with on column DNase I digestion (Qiagen), and RNA from fibroblast was extracted with Trizol regent first (Thermo fisher), then purified with RNeasy kit with on column DNase I digestion. High MW Northern was performed as described. 10 ug RNA samples were treated in 30 ul denaturing solution for 2 min at 80°C (deionized formamide: 13.125 ul; 40% formaldehyde: 3.75 ul; 10X MOPS buffer: 3 ul; 2 mg/ml EtBr: 0.375 ul; Formamide loading dye: 1 ul), then put on ice.
  • RNA samples were run in agarose gel containing 7% formaldehyde with 1XMOPS buffer containing 0.7% formaldehyde. 100V for 1 h, then 250V 2h, +/- buffer was changed every 30 min. Gel was washed 3 times with DEPC-treated water, then treated in gel denaturing buffer containing 50mM NaOH and1 .5 mM NaCI for 30min. After washed by 20X SSC for 45 min, gel was semi-dry transferred to nylon membrane in 10XSSC O.N. Membrane was rinsed in 2XSSC, crosslinked by 125 mJ UV, then baked at 80 °C for 1 hour.
  • Hybridization was performed in Ultrahyb-oligo buffer with 32 P-Y[ATP]-labeled (AATCCC) 4 oligo at 42°C O.N. Membrane was washed by 2X SSC 0.1%SDS RT for 10 min, then 2X SSC 0.5%SDS at 42V for 30 min. For dot-blotting, 5 pg RNA was denatured by adding equal volume of Northern-Max Gly sample loading dye (Invitrogen) at 50°C for 1 hour, then slot blot to Hybound-N + . Ultrahyb-oligo buffer with 3 2P-y[ATP]-labeled (AATCCC) 4 oligo was used for hybridization at 42°C for 48 hours. Membranes were washed twice with 2XSSC 0.1%SDS and exposed to phosphor image. The radioactive signal was detected by phosphor image.
  • RNA was used for reverse transcription using superscript III (Thermo Fisher) with (AATCCC)e and GAPDH primer. Chromosome-specific TERRA quantification was performed following the published protocol on Thermo Fisher QS3 [Feretzaki M, 2017] .
  • TERRA transcripts displayed big length heterogeneity [Azzalin CM, 2007].
  • the first correlation between TERRA length and telomere length was reported in fibroblasts derived from patients with ICF syndrome (Immunodeficiency, Centromeric region instability, Facial anomalies).
  • the elongation of telomeres caused by overexpression of telomerase is accompanied with increase in the length of TERRA transcripts [Yehezkel S, 2008]. It was further proved that TERRA length is dependent on telomere length using various human cell lines with over-elongated telomere caused by ectopic telomerase overexpression [Arnoult N, 2012; Van Beneden A, 2013],
  • ALT cells have highly heterogeneous, fluctuating telomere lengths [Bryan, T.M., 1995], and high levels of telomere sister chromatid exchanges (t-SCEs) [Londono-Vallejo, J. A., 2004], abundant extrachromosomal telomeric repeat DNA (ECTR) [Cesare, A. J, 2004; Nabetani, A., 2009].
  • TERRA telomere sister chromatid exchanges
  • supt4 siRNA Various concentrations of supt4 siRNA were used to knock down Supt4 abundance in U2OS cells for 5 days and 10 days respectively (FIG. 8), then dot-blotting was used to measure the UUAGGG repeat abundance of TERRA transcript in the RNA sample. As shown in FIG. 1A and quantified in FIG. 1 B, UUAGGG repeat abundance in all the U2OS cells treated with supt4 siRNA was decreased up to 90% compared with the pooled siRNA control (FIG. 1 A & 1 B, supt4 KN vs siRNA Ctrl).
  • TERRA are essential for the proliferation of ALT + cancer cells, since TERRA enables DNA-RNA hybrids “R loop” formation at chromosome ends which can promote homologous recombination among telomeres to sustain genome instability and avoid cellular senescence [Arora, R., 2014], The fact that supt4 KN decreases long TERRA repeat abundance indicates the depletion of supt4 function may be connected to inhibition of alternative lengthening of telomere (ALT) in U2OS cells.
  • CCCTAA Partially single-stranded telomeric n circle (C-circles) generated by telomere recombination are specific and quantifiable markers of ALT [Grudic, A., 2007]
  • C circle assay CO assay
  • ALT tumors which is the rolling circle amplification of partially double stranded C-circles by 29 DNA polymerase which is auto-primed by the partial G-strand ([TTAGGG] n ), producing long telemetric ssDNA concatemers which can be detected by dot-blotting [Henson, J., 2009].
  • the result of CC assay in FIG. 3A shows Ienti-supt4 knock-down (FIG.
  • Supt4 KN inhibits the proliferation of both ALT' and ALT + cancer cells.
  • the cell numbers of supt4 siRNA treated U2OS was counted after 5 days and 10 days of incubation (FIG. 4A, FIG. 9), and dose-dependent decrease was detected. Moreover, 10 days supt4 siRNA treatment causes more significant effect compared with 5 days treatment. It is consistent with the data in FIG. 1 -3 that supt4 knock down inhibits the ALT in U2OS cells, which is essential for the cell proliferation.
  • Hela is an immortal cervical cancer cell line which have an active telomerase to maintain the rapid cell growth [Ivankovic, M., 2007], and there is no observed ALT in Hela cell, therefore, Hela is considered as ALT". Similar growth inhibitory effect was observed in Hela cells (FIG. 4B), indicating there is more targets of supt4 function in cancer cells.
  • telomere Since maintain telomere is essential for cancer cell survival, FACS was used to determine whether supt4 depletion in U2OS can lead to apoptosis in this ALT+ cancer cells.
  • the immortal cervical cancer cell line Hela (ALT-) was used as the negative control, since it harbors an active telomerase to maintain the rapid cell growth [Ivankovic, M., 2007], After being treated with 10nM supt4 siRNA for 10 days, the cells were stained with two dyes: Annexin V stains apoptotic cells by its ability to bind to phosphatidylserine, and PI stains dead cells.
  • Recombinant Annexin V is conjugated to Alexa Fluor 488, which is a perfect spectral match to fluorescein (FITC).
  • FITC fluorescein
  • telomere is an immortal cervical cancer cell line which relies on active telomerase to maintain the rapid cell growth [Ivankovic, M., 2007], It is possible that Supt4 knock down suppresses telomerase activity to cause growth delay.
  • Telomerase is a ribonucleoprotein responsible for maintaining telomere length.
  • the core of telomerase has two components: Catalytic telomerase reverse transcriptase (TERT) and telomerase RNA (TERC).
  • TERT utilizes the template region (3’-CAAUCCCAAUC-5’) of TERC to add TTAGGG DNA repeats and thereby extend single stranded 3' telomeric strands [Morin, G.B., 1989].
  • telomerase cajal body protein 1 TCAB1
  • H/ACA-motif RNA binding proteins dyskerin NHP2, NOP10, Gar1
  • ATPase proteins Pontin and Reptin [Xu, Y bracket 2016].
  • telomerase activity of Hela was tested by TRAP assay combined with real-time analysis (TRAPeze-RT), as well as telomerase activity of ALT U2OS and normal fibroblast BJ (FIG. 7A).
  • the result shows Hela harbors very strong telomerase activity compared with the normal fibroblasts which has no active telomerase (FIG. 7A).
  • Supt4 knock down suppresses telomerase activity in Hela to 50% (FIG. 7B), which is consistent with the inhibition of its proliferation caused by supt4 Knock-down (FIG.
  • TERRA gets transcribed from the telomeric C-rich strand and therefore harbors sequences that are complementary to the RNA subunit (template region) of telomerase.
  • TERRA acts as a potent in vitro inhibitor of telomerase, in part, by directly base pairing to the telomerase RNA moiety [Redon, S., 2010].
  • recent publications suggest that in vivo TERRA may positively affect telomerase function.
  • TERRA might assist telomere lengthening in telomerase positive cells by recruiting telomerase to the shortest telomeres in cells [Moravec M, 2016].
  • TERRA levels in cancers could simultaneously suppress telomerase-mediated and ALT-mediated telomere elongation and therefore progression of different cancer types. [Cusanelli E, 2013]. And interestingly, Alternative Lengthening of Telomeres can coexist with active Telomerase [Perrem, K., 2001],
  • qRT-PCR measurement can detect low abundant TERRA and make the accurate quantification of the numbers of TERRA molecules, but the signals are from short sequences in the subtelomeric region of TERRA transcript, it could't be used for the analysis for TERRA length regulation.
  • Porro et al. reported the mUUAGGG tract-specific reverse transcription (Porro A, 2010).
  • Cellular RNA was polyadenylated in vitro with recombinant yeast Papl, then was reverse transcribed with a mixture of six oligonucleotides that were predicted to hybridize to the junction between the UUAGGG sequence in all six permutated registers and the poly(A).
  • RT was first carried out in the presence of only three nucleotides ([32P]dCTP, dTTP, and dATP) in order to restrict reverse transcription to the pure UUAGGG tract.
  • the 1-h pulse was followed by a chase reaction in which an excess of unlabeled dCTP and, for the dGTP reaction, dGTP was added in order to allow RT beyond the cytosine-containing subtelomeric TERRA sequence.
  • dGTP was added in order to allow RT beyond the cytosine-containing subtelomeric TERRA sequence.
  • This RT method to detect the length differences of TERRA repeat in supt4 siRNA treated cells.
  • the positive control was successful, showing the majority of the shorter products were chased into longer cDNAs with dGTP, indicating dGTP is required to reverse transcribe beyond cytosine bases (data not shown).
  • ALT cancers utilize BIR (break-induced replicate ALT mechanisms on) -based mechanism for telomere elongation and are usually associated with poorer prognosis.
  • BIR break-induced replicate ALT mechanisms on
  • TERRA an RNA transcribed using telomeric DNA as a template.
  • the DNA template consists at least in part of repeats of a DNA sequence six nucleotides in length.
  • TERRA is a crucial factor in triggering BIR and promoting telomere elongation in certain types of cancer.
  • SUPT4H1 a gene that encodes one of the DSIF (DRB sensitivity-inducing factor) elongation factor subunits, is required for the transcription of TERRA in ALT-dependent cancer cells and interference with Spt4h production can decrease TERRA transcript production and length, and decrease of Supt4h can lead to shortening of telomeres in cancer cells grown in culture. Associated with telomere shortening during cell culture is increased apoptotic cell death.
  • DSIF DRB sensitivity-inducing factor
  • telomere length involved telomeric DNA containing nucleotide repeats.
  • SUPT4H1/5H a highly conserved transcription elongation complex, can regulate RNA polymerase II processivity.
  • SUPT4H1 has been reported as one of the key factors for the expression of long trinucleotide repeat containing transcripts in multiple disease models. Our investigations disclosed here indicate that SUPT4H1 is required for efficient transcription of TERRA, a hexanucleotide repeat (TTAGGG) 6 non-coding RNA transcribed from the chromosome telomere region.
  • TTAGGG hexanucleotide repeat
  • SUPT4H1 may be a target gene in treating ALT cancers.
  • telomerase activity in non-ALT cancer cells is reduced relative to total protein and also relative to nonspecific protein controls by interference with Supt4h. It thus appears that reduced Sujpt4h may interfere with the telomerase preservation of telomere length. As such, Supt4h functioning in transcription through nucleotide repeats of telomeres may be a target also for treatment of non-ALT cancers because of Supt4h effects on telomerase activity.
  • RNATERRA a noncoding RNA connecting telomere biology to genome integrity.
  • RNA TERRA is induced by telomere shortening to nucleate telomerase molecules at short telomeres.
  • TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe.
  • RNA TERRA is a natural ligand and direct inhibitor of human telomerase.

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Abstract

Methods of treating a subject for a cellular proliferative disease, e.g., a cancer, are provided. Aspects of the methods include: administering to the subject an agent that modulates DSIF complex activity, e.g., activity of a DSIF complex made up of a SPT4 and SPT5 protein, such as a DSIF complex made up of Supt4h and Supt5h, in a manner sufficient to treat the subject for the cellular proliferative disease. Also provided are compositions for practicing the methods.

Description

TREATMENT OF CELLULAR PROLIFERATIVE DISEASE VIA DSIF COMPLEX MODULATION, AND COMPOSITIONS FOR PRACTICING THE SAME
CROSS-REFERENCE TO RELATED APPLICATION
Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63/408,672 filed September 21 , 2022; the disclosure of which application is incorporated herein by reference.
INTRODUCTION
Transcription elongation factor Spt4 was first identified in Saccharomyces cerevisiae [Winston, F., 1984], and is highly conserved among eukaryotes [Guo, M. 2008; Hartzong, G.A., 1996; Wenzel, B.M., 2010]. The DRB sensitivity-inducing factor (DSIF) complex formed by Spt4 and Spt5 has been implicated in multiple steps of RNAPII transcription, including promoter proximal pausing, elongation, and RNA processing/termination [Hartzog, 2013]. Spt4 enhances RNA polymerase II processivity by reducing dissociation of polymerase from highly structured regions of DNA template, most frequently in the repeat region. [Rondon, A.G., 2003; Mason, P.B., 2005; Hirtreiter, A., 2010]. In the recent years, the 14 KDa human ortholog Supt4 has been shown to be required to facilitate the transcription of pathogenic RNA with abnormally long trinucleotide repeats in multiple neurodegenerative diseases, such as Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS) [Kramer NJ, 2016; Cheng HM, 2015; Liu CR, 2012], In a disease model for HD, depleting Spt4 reduced HTT RNA/protein expression and toxicity associated with long (CAG) repeats in yeast and cultured neurons [Liu, 2012], Mechanistic studies revealed the repeat-length specificity of Spt4 regulation, as Spt4 deletion reduced RNAPII occupancy downstream of a (CAG)gg expansion, whereas this effect was not observed with (CAG)2- repeat. Further work showed that downregulation of mouse Spt4 in (CAG)ns HD-mouse model reduced HTT RNA/protein expression in vivo and was associated with reduced neurodegenerative effects [Cheng HM, 2015].
Telomeres are nucleoprotein complexes that protect the physical ends of linear chromosomes from degradation and unscheduled repair activities [Lingner, J., 1995; Sfeir, A. 2012], Telomeres were considered transcriptionally silent regions of mammalian chromosomes until Azzalin et al. discovered that telomeres are transcribed into long noncoding RNA known as Telomeric Repeat-containing RNA (TERRA) in 2007 [Azzalin, C.M., 2007], TERRA molecules are comprised of subtelomere and telomere-derived UUAGGG repeats and vary in length from 100 bases to 9 kb in mammals [Azzalin, C.M., 2007], For the past decade, the structure and function of TERRA molecules, as well as their transcriptional regulation and their importance in the maintenance of telomeric lengths have been investigated. TERRA has been proved to play critical roles in telomere biology, including regulation of telomere length, telomerase activity and heterochromatin formation at chromosome ends [Cusanelli, E., 2013].
TERRA transcription initiates from subtelomeric promoters located on at least two- thirds of chromosome ends [Nergadze, S., 2009; Deng Z, 2012; Porro A, 2014], Those subtelomeric promoters comprise CpG dinucleotide-rich DNA islands, characterized by 61 - 29-37 repeats upstream of TERRA Transcription Start Site (TSS) and at ~ 1 kb from the telomeric repeat region [Brown WRA, 1990; Nergadze, 2009]. Active transcription of CpG promoters was proved by the binding of RNA polymerase II to both 61-29-37 repeats and telomeric repeats [Nergadze, 2009] and the interactions of CTCF transcription factor and cohesin with human subtelomeres [Deng Z, 2012], Other reported transcription regulators of TERRA include ATRX chromatin remodelers [Eid,R., 2015], cohesin subunit Rad21 [Episkopou,H., 2014], nuclear respiratory factor 1 (NRF1 ) [Diman, A, 2016], Rb [Gonzalez- Vasconcellos, I., 2017], and SnaiH via P53 [Mazzolini R, 2018].
Since TERRA harbors long UUAGGG repeats (corresponding to telomeric TTAGGG repeats) transcribed from highly structured telomere regions, it is speculated that the elongation activity of RNA polymerase II could regulate TERRA transcription. Recently, a transcription elongation factor has been identified as a new regulator of TERRA in yeast [Rodrigues J, 2018]. Paf1 and Ctr9, two components of yeast transcription elongation complex PAF1 , help to maintain low TERRA levels, and further analysis of Paf1 and Ctr9 double mutants showed that high TERRA levels are correlated with poor fitness through mechanisms that are independent of previously identified regulators of TERRA such as Sir4, Rati , and Trf4. Another recently published work showed specific genetic interaction between the specialized telomeric CST complex (Cdc13, Stn1 and Ten1) and Spt4 and Spt5 in Saccharomyces cerevisiae [Calvo O, 2019]. Moreover, the CST complex was found to physically associate with Spt5 and regulate its association with chromatin during active transcription, indicating that Spt5 is a target of CST in transcription regulation. However, there is no report of regulation of TERRA transcription elongation in mammalian cells.
SUMMARY
Methods of treating a subject for a cellular proliferative disease, e.g., a cancer, are provided. Aspects of the methods include: administering to the subject an agent that modulates DSIF complex activity, e.g., activity of a DSIF complex made up of a SPT4 and SPT5 protein, such as a DSIF complex made up of Supt4h and Supt5h, in a manner sufficient to treat the subject for the cellular proliferative disease. Also provided are compositions for practicing the methods.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Supt4 siRNA knock-down decreases TERRA repeat abundance in U2OS cells.
U2OS cells were treated with 20nM, 6.6nM, 2.2nM supt4 siRNA and control siRNA for 5 days and 10 days respectively, RNA was extracted and analyzed. (A) TERRA repeat abundance were analyzed by dot blot using 32P-y[ATP]-labeled probe generated by endlabeling of (AATCCC)4 oligo. The levels of input RNA were normalized to 18S RNA. (B) Quantification analysis of the dot-blotting results in (A). (C) Quantitative real-time analysis of abundance of subtelomere sequences from individual chromosome specific TERRA in U2OS cells treated with 20nM Supt4 siRNA for 10 days.
Figure 2. Supt4 siRNA knock-down decreases long TERRA transcript in U2OS cells.
(A) U2OS cells were treated with 10nM supt4 siRNA and control siRNA for 10 days respectively, RNA was extracted and analyzed by 32P-y[ATP]-labeled TERRA probe by high MW Northern as described in experimental procedures. The levels of input RNA were normalized to 18S RNA. (B) Quantification analysis was performed in Typhoon software.
Figure 3. Ienti-Supt4 knock-down decreases C-circle formation and telomere repeat abundance in ALT+ U2OS cells.
U2OS cells were transfected with Ienti-supt4 shRNA, and control empty virus with puromycin resistance (puro Ctrl) respectively for 20 days, genomic DNA was extracted and analyzed by C-circle formation (A) and Quantitative real-time PCR analysis of telomere repeat abundance (B). The levels of Supt4 transcript and protein in U2OS cells treated with Ienti-supt4 and puro control virus were detected in (C) and (D), GAPDH was used as internal control.
Figure 4. The size and abundance of telomeres in U2OS cells treated with lenti- supt4 determined by genomic TRF assay. (A) U2OS cells were transfected with Ienti-supt4 shRNA, and control empty virus with puromycin resistance (puro Ctrl) respectively for 20 days, genomic DNA was extracted and analyzed by Southern-TRF with 32P-Y[ATP]-labeled telomere G probe. (B) Quantification of the signal in high MW region and low MW region was performed in Typhoon software.
Figure 5. Supt4 siRNA knock-down increases cell apoptosis in ALT+ U2OS cells, but not in ALT’ Hela cells. U2OS cells (A) and Hela cells (B) were treated with 10nM supt4 siRNA for 10 days before cells were collected for FACS analysis. Alexa Fluor 488 Annexin V/Dead cell apoptosis kit was used to analyze the status of each cell. Cells were stained with two dyes: Annexin V stains apoptotic cells by its ability to bind to phosphatidylserine, and PI stains dead cells. Recombinant Annexin V is conjugated to Alexa Fluor 488, which is a perfect spectral match to fluorescein (FITC). (Q1 : PI-, FITC+: apoptotic cells; Q2: PI+, FITC+: late apoptotic cells; Q3: PI+, FITC-: necrotic cells; Q4: PI-, FITC-: viable cells).
Figure 6. The effects of Supt4 siRNA knock-down on TERRA repeat abundance in SAOS-2 and MG-63 compared with U2OS.
(A) SAOS-2, MG-63, and U2OS cells were treated with 10 nM supt4 siRNA and control siRNA for 10, RNA was extracted and TERRA repeat abundance were analyzed by dot blot using 32P-Y[ATP]-labeled probe generated by end-labeling of (AATCCC)4 oligo. The levels of input RNA were normalized to 18S RNA. (B) Supt4 transcript abundance in the various knock-down strains.
Figure 7. Supt4 siRNA knock-down decreases telomerase activity in ALT- Hela cells. Quantitative TRAPeze-RT analysis of telomerase activity using CHAPS extracted cellular protein extract. Ct. values of real-time reaction were converted into TPG units using a standard curve generated by dilution series of telomerase substrate oligonucleotide TSR8 with eight telomeric repeats. A TSV template was used as internal control of PCR amplification. Heated samples served as the negative control for telomerase (labeled as with heat). (A) Relative telomerase activity of normally growing Hela (ALT-), U2OS (ALT+), and BJ (fibroblast) cells. (B) Relative telomerase activity of Hela cells treated with supt4 siRNA for 5 days.
Figure 8. Supt4 RNA and protein abundance in U2OS cells treated with supt4 siRNA. U2OS cells were treated with 20 nM, 6.6 nM, 2.2 nM supt4 siRNA and control siRNA for 5 days and 10 days respectively. (A) RNA was extracted and analyzed for the relative levels of Supt4 transcript. GAPDH was used as internal control. (B) Protein samples from U2OS cells treated with 6.6 nM supt4 siRNA for 5 and 10 days were analyzed by Western-blotting for Supt4 protein abundance.
Figure 9. 72 hours Supt4 siRNA knock-down has no effect on ALT in U2OS cells. U2OS cells were treated with 10nM supt4 siRNA, control siRNA, and lipofectamine RNAimax (lipo) respectively for 72 hours, genomic DNA was extracted and analyzed. (A) Genomic TRF assay to determine the size and abundance of telomeres. (B). Quantitative real-time PCR analysis of telomere repeat abundance. (C) Detection of C-circles by CC assay using serial dilutions (1/3) of reaction products. (D) The relative levels of Supt4 transcript in U2OS cells treated with supt4 siRNA and control siRNA for 72 hours. GAPDH was used as internal control. Figure 10. Supt4 RNA abundance in SAOS-2, MG-63, and U2OS cells.
SAOS-2, MG-63, and U2OS cells were growing in DMEM+10%FBS. RNA was extracted and analyzed for the relative levels of Supt4 transcript. GAPDH was used as internal control. U2OS cells were transfected with Ienti-supt4 shRNA, and control empty virus with puromycin resistance (puro Ctrl) respectively for 20 days. RNA was extracted and analyzed for the relative levels of Supt4 transcript. GAPDH was used as internal control. - Protein were analyzed by Western-blotting for Supt4 protein abundance.
Figure 11. Effect of supt4 siRNA KN on C circle formation in U2OS and SAOS- 2. SAOS-2 and U2OS cells were treated with 10 nM supt4 siRNA and control siRNA for 5 days and 10 days respectively. DNA was extracted and analyzed for C-circles by CO assay using serial dilutions (1/3) of reaction products.
Figure 12. HD143 treatment affects the proliferation of U2OS and MG-63. The cells were treated with different doses of HD143 and its derivative CPD25 for 72 hours. Titer-glow assay was used to measure the viable cells after the compound treatment.
Figure 13. Supt4 siRNA knock-down inhibits the growth of Hela (ALT ) cells.
Hela cells were treated with 10nM, 2 nM, 0.4 nM supt4 siRNA and control siRNA respectively for 5 days and 10 days in 10cm culture plate. The total cell number was counted by vi-cell.
DETAILED DESCRIPTION
Methods of treating a subject for a cellular proliferative disease, e.g., a cancer, are provided. Aspects of the methods include: administering to the subject an agent that modulates DSIF complex activity, e.g., activity of a DSIF complex made up of a SPT4 and SPT5 protein, such as a DSIF complex made up of Supt4h and Supt5h, in a manner sufficient to treat the subject for the cellular proliferative disease. Also provided are compositions for practicing the methods.
Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
METHODS
Aspects of the present disclosure include methods for treating a subject for a cellular proliferative disease. Cellular proliferative diseases are conditions characterized by abnormal cellular proliferation, such as cancers. Such disease conditions include cancer and neoplastic diseases and other diseases characterized by the presence of unwanted cellular proliferation, e.g., hyperplasias, and the like. In certain embodiments, the cellular proliferative disease is a cancer, and in some instances is an ALT+ cancer.
In embodiments of the method, the methods include administering to the subject an agent that modulates DSIF complex activity in a manner sufficient to treat the subject for the cellular proliferative disease. The target DSIF complex in such embodiments may vary, where in some embodiments the target DSIF complex comprises an SPT4 protein and an SPT5 protein, such as a DSIF complex comprising Supt4h and Spt5h.
Depending on the particular embodiments being practiced, a variety of different types of active agents may be employed. Active agents of interest include, but are not limited to, small molecules, nucleic acids (e.g., DNA, RNA), proteins (e.g., antibodies, enzymes, nucleases, proteases, etc.), and the like, or any combination thereof. In some instances, the agent modulates the activity of the protein following expression, such that the agent is one that changes the activity of the protein encoded by the target gene following expression of the protein from the target gene. In these instances, the agent is one that may act directly with protein encoded by the target gene. In these instances, the agent may be one that selectively reduces the deleterious activity, e.g., transcription of extremely long repeat regions in non-genic DNA, of the encoded protein, but retains or enhances, at least to a detectable level, the beneficial activity of the encoded protein. In certain embodiments, such agents are not inhibitors of the protein, but instead selectively reduce the deleterious activity of the protein via another mechanism, e.g., by reducing the amount of the protein in the cell that is available for interaction with other proteins, by reducing production of the protein, , by preventing assembly of a protein complex, etc.
In yet other embodiments, the agent modulates expression of the RNA and/or protein from the gene, such that it changes the expression of the RNA or protein from the target gene in some manner. In these instances, the agent may change expression of the RNA or protein in a number of different ways.
In some embodiments, DSIF complex modulating agents of interest include selective SPT4 modulatory agents. Selective SPT4 modulatory agents are agents that selectively change the SPT4 activity in a cell, e.g., decrease SPT4 activity in a cell. In some instances, the target SPT4 activity that is modulated, e.g., decreased, by the active agent is a transcription activity, and specifically an activity that facilitates RNA polymerase II processivity through target repeat domains, e.g., long TTAGGG repeat domains. The target SPT4 activity that is modulated by such agents is an activity arising from an SPT4 protein. The term SPT4 protein is used herein to collectively refer to not only yeast Spt4 proteins, but also mammalian homologs thereof, e.g., human SUPT4H; murine Supt4h, etc. As such, SPT4 proteins of interest whose activity may be modulated by the selective SPT4 modulatory agents include, but are not limited to, S. cerevisiae Spt4; human SUPT4H and murine Supt4h.
Where the agent employed in methods of the invention is an SPT4 modulatory agent, the modulatory agent that is employed may be any agent that, upon introduction into a cell, changes the SPT4 activity of the cell, and specifically reduces the SPT4-mediated extended target repeat transcription activity in the subject. The SPT4 modulatory agent may modulate activity in a number of different ways, e.g., by reducing expression of an SPT4 protein, by inhibiting binding of an SPT4 protein to another protein, e.g., a protein interacting with SPT4 (e.g., an SPT5 protein, such as Supt5h (Spt5 or SUPT5H)), by preventing SPT4 from forming a DSIF complex with SPT5, etc. Examples of different types of modulatory agents are now reviewed in greater detail below.
In certain embodiments, the agent is one that reduces, including inhibits, expression of a functional SPT4 protein. Inhibition of SPT4 protein expression may be accomplished using any convenient means, including use of an agent that inhibits SPT4 protein expression, such as, but not limited to, antisense agents, RNAi agents, agents that interfere with transcription factor binding to a promoter sequence of the SPT4 gene, or inactivation of the SPT4 gene, e.g., through recombinant techniques, etc.
For example, antisense molecules can be used to down-regulate expression of an SPT4 gene in the cell. The anti-sense reagent may be antisense oligodeoxynucleotides (ODN), particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA. The antisense sequence is complementary to the mRNA of the targeted protein, allowing the antisense sequence to hybridize to the mRNA and form a duplex that inhibits expression of the targeted protein. Antisense molecules inhibit gene expression through various mechanisms, e.g., by reducing the amount of mRNA available for translation, through cleavage of the RNA:DNA heteroduplex by activation of RNAse H, or steric hindrance. One or a combination of antisense molecules may be administered, where a combination may include multiple different sequences. Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the transcriptional initiation is oriented such that an antisense strand is produced as an RNA molecule. Alternatively, the antisense molecule is a synthetic oligonucleotide. Antisense oligonucleotides will generally be at least about 7, usually at least about 12, more usually at least about 20 nucleotides in length, and not more than about 500, usually not more than about 50, more usually not more than about 35 nucleotides in length, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like. It has been found that short oligonucleotides, of from 7 to 8 bases in length, can be strong and selective inhibitors of gene expression (see Wagner et al. (1996), Nature Biotechnol. 14:840-844).
Targets for antisense molecules may be a specific region or regions of the endogenous sense strand mRNA sequence that are chosen to be complemented by the antisense sequence. Selection of a specific sequence for the oligonucleotide may use an empirical method, where several candidate sequences are assayed for inhibition of expression of the target gene in an in vitro or animal model. A combination of sequences may also be used, where several regions of the mRNA sequence are selected for antisense complementation.
Antisense oligonucleotides may be chemically synthesized by methods known in the art (see Wagner et al. (1993), supra, and Milligan et al., supra.) Oligonucleotides may be chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases.
Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-0'-5'-S-phosphorothioate, 3'-S-5'-0-phosphorothioate, 3'-CH2-5'-O- phosphonate and 3'-NH-5'-0-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity. The a-anomer of deoxyribose may be used, where the base is inverted with respect to the natural (3-anomer. The 2'-OH of the ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl sugars, which provides resistance to degradation without comprising affinity. Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl- 2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. 5- propynyl-2'- deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.
As an alternative to anti-sense inhibitors, catalytic nucleic acid compounds, e.g. ribozymes, anti-sense conjugates, etc. may be used to inhibit gene expression. Ribozymes may be synthesized in vitro and administered to the patient, or may be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell (for example, see International patent application WO 9523225, and Beigelman et al. (1995), Nucl. Acids Res. 23:4434-42). Examples of oligonucleotides with catalytic activity are described in WO 9506764. Conjugates of anti-sense ODN with a metal complex, e.g. terpyridylCu(ll), capable of mediating mRNA hydrolysis are described in Bashkin et al. (1995), Appl. Biochem. Biotech not. 54:43-56.
In addition, the transcription level of a SPT4 protein can be regulated by gene silencing using RNAi agents, e.g., double-strand RNA (Sharp (1999) Genes and Development 13: 139-141). RNAi, such as double-stranded RNA interference (dsRNAi) or small interfering RNA (siRNA), has been extensively documented in the nematode C. elegans (Fire, A., et al, Nature, 391 , 806-811 , 1998) and routinely used to “knock down” genes in various systems. RNAi agents may be dsRNA or a transcriptional template of the interfering ribonucleic acid which can be used to produce dsRNA in a cell. In these embodiments, the transcriptional template may be a DNA that encodes the interfering ribonucleic acid. Additional RNAi mediators include shRNA, microRNAs (miRNA), and artificial miRNAs. Methods and procedures associated with RNAi are also described in WO 03/010180 and WO 01/68836, all of which are incorporated herein by reference. dsRNA can be prepared according to any of a number of methods that are known in the art, including in vitro and in vivo methods, as well as by synthetic chemistry approaches. Examples of such methods include, but are not limited to, the methods described by Sadher et al. (Biochem. Int. 14:1015, 1987); by Bhattacharyya (Nature 343:484, 1990); and by Livache, et al. (U.S. Patent No. 5,795,715), each of which is incorporated herein by reference in its entirety. Single-stranded RNA can also be produced using a combination of enzymatic and organic synthesis or by total organic synthesis. The use of synthetic chemical methods enables one to introduce desired modified nucleotides or nucleotide analogs into the dsRNA. dsRNA can also be prepared in vivo according to a number of established methods (see, e.g., Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed.; Transcription and Translation (B.D. Hames, and S.J. Higgins, Eds., 1984); DNA Cloning, volumes I and II (D.N. Glover, Ed., 1985); and Oligonucleotide Synthesis (M.J. Gait, Ed., 1984, each of which is incorporated herein by reference in its entirety). A number of options can be utilized to deliver the dsRNA into a cell or population of cells such as in a cell culture, tissue, organ or embryo. For instance, RNA can be directly introduced intracellularly. Various physical methods are generally utilized in such instances, such as administration by microinjection (see, e.g., Zernicka-Goetz, et al. (1997) Development 124:1133-1137; and Wianny, et al. (1998) Chromosoma 107: 430-439). Other options for cellular delivery include permeabilizing the cell membrane and electroporation in the presence of the dsRNA, liposome-mediated transfection, or transfection using chemicals such as calcium phosphate. A number of established gene therapy techniques can also be utilized to introduce the dsRNA into a cell. By introducing a viral construct within a viral particle, for instance, one can achieve efficient introduction of an expression construct into the cell and transcription of the RNA encoded by the construct.
In another embodiment, the SPT4 gene is inactivated so that it no longer expresses a functional protein. By inactivated is meant that the gene, e.g., coding sequence and/or regulatory elements thereof, is genetically modified so that it no longer expresses a functional SPT4 protein, e.g., at least with respect to SPT4 transcription activity through a region of extended target repeats. In some instances, expression of the SPT4 protein may be reduced or inhibited. In some cases, a non-functional (e.g., mutated) SPT4 protein may be expressed.
The alteration or mutation may take a number of different forms, e.g., through deletion of one or more nucleotide residues, through exchange of one or more nucleotide residues, and the like. Various methods of gene editing may be employed, including e.g., those methods capable of introducing a single nucleotide substitution, those methods capable of introducing a site-directed deletion and those methods capable of introducing site-directed insertion. Useful site-directed gene editing methods, described in more detail below, include methods that employ a nuclease to cleave one or both strands of a target nucleic acid molecule. The cleaved target nucleic acid may be subsequently repaired, e.g., through homology directed repair (HDR), to introduce the edit at a desired location. Methods for generating targeted gene modifications through homologous recombination are known in the art, including those described in: U.S. Patent Nos. 6,074,853; 5,998,209; 5,998,144; 5,948,653; 5,925,544; 5,830,698; 5,780,296; 5,776,744; 5,721 ,367; 5,614,396; 5,612,205; the disclosures of which are herein incorporated by reference.
Many gene editing systems, including e.g., Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)Znuclease systems, Transcription Activator-Like Effector Nuclease (TALEN) systems, Zinc Finger Nuclease (ZFN) systems, and the like, are well developed and can produce single or double strand-breaks with high fidelity. Methods of site-directed introduction of a desired edit will vary and may include introducing a site directed cleavage event, e.g., through the use of a site-directed nuclease (e.g., a CRISPR/Cas9 nuclease, a TALEN nuclease, a ZFN, and the like) followed by a specific repair event at the site cleaved by the site-directed nuclease. Such methods of specific repair may include, e.g., homologous recombination, including homology directed repair (HDR). Methods for gene editing using gene editing systems are known in the art (see, e.g., Gratz et al. (2014) Genetics. 196(4)961-971 ; Chu et al. (2015) Nature. 33:543-548; Hisano et al. (2015) Scientific Reports 5: 8841 ; Farboud & Meyer (2015) Genetics, 199:959-971 ; Merkert & Martin (2016) Stem Cell Research 16(2):377-386; Urnov et al. (2005) Nature. 435(7042) :646-5; Beumer et al (2006) Genetics. 172(4):2391-2403; Meng et al (2008) Nat Biotechnol. 26(6):695-701 ; Perez et al. (2008) Nat Biotechnol. 26(7):808-816; Hockemeyer et al. (2009) Nat Biotechnol. 27(9):851-7; Zu et al. (2013) Nature Methods. 10:329-331 ; Cui et al. (2015) Scientific Reports 5:10482; Liu et al. (2012) J. Genet. Genomics. 39:209-215, Bedell et al. (2012) Nature. 491 :114-118, Wang et al. (2013) Nat. Biotechnol. 31 :530-532; Ding et al. (2013) Cell Stem Cell. 12:238-251 ; Wefers et al. (2013) Proc. Natl. Acad. Sci. U. S. A., 110:3782-3787; the disclosures of which are incorporated herein by reference in their entirety).
Also of interest in certain embodiments are dominant negative mutants of SPT4 proteins, where expression of such mutants in the cell result in a modulation, e.g., decrease, in SPT4 mediated transcription of extended target repeats in a cell. Dominant negative mutants of SPT4 are mutant proteins that exhibit dominant negative SPT4 activity. As used herein, the term "dominant-negative SPT4 activity" or "dominant negative activity" refers to the inhibition, negation, or diminution of certain particular activities of SPT4, and specifically to SPT4 mediated transcription of extended target repeats. Dominant negative mutations are readily generated for corresponding proteins. These may act by several different mechanisms, including mutations in a substrate-binding domain; mutations in a catalytic domain; mutations in a protein binding domain (e.g. multimer forming, effector, or activating protein binding domains); mutations in cellular localization domain, etc. A mutant polypeptide may interact with wild-type polypeptides (made from the other allele) and form a non-functional multimer. In certain embodiments, the mutant polypeptide will be overproduced. Point mutations are made that have such an effect. In addition, fusion of different polypeptides of various lengths to the terminus of a protein, or deletion of specific domains can yield dominant negative mutants. General strategies are available for making dominant negative mutants (see for example, Herskowitz (1987) Nature 329:219, and the references cited above). Such techniques are used to create loss of function mutations, which are useful for determining protein function. Methods that are well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriptional and translational control signals for increased expression of an exogenous gene introduced into a cell. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Alternatively, RNA capable of encoding gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, M. J. ed., IRL Press, Oxford.
In yet other embodiments, the agent is an agent that modulates, e.g., inhibits, SPT4 activity by binding to SPT4 and/or inhibiting binding of SPT4 to a second protein, e.g., an SPT5 protein, such as Spt5 or SUPT5H. The term SPT4 protein is used herein to collectively refer to not only yeast Spt4 proteins, but also mammalian homologs thereof, e.g., human SUPT4H; murine Supt4h, etc. As such, SPT4 proteins of interest whose activity may be modulated by the selective SPT4 modulatory compounds include, but are not limited to, S. cerevisiae Spt4; human SUPT4H and murine Supt4h. The subject compounds may be referred to as SPT4 modulatory agents. SPT4 modulatory agents are compounds that change the SPT4 activity in a cell, e.g., decrease SPT4 activity in a cell. The compound may be a selective SPT4 modulatory agent. In some instances, the target SPT4 activity that is modulated, e.g., decreased, by the active compound is a transcription activity, and specifically an activity that facilitates RNA polymerase II processivity through a target repeat. The target SPT4 activity that is modulated by such compounds is an activity arising from an SPT4 protein.
Where the subject compound employed in methods of the invention is an SPT4 modulatory agent, the compound that is employed may, upon introduction into a cell, change the SPT4 functionality in the cell, and at least differentially reduce the SPT4 mediated extended target repeat transcription activity in the subject. The SPT4 modulatory agent may modulate functionality in a variety of ways, e.g., by inhibiting binding of an SPT4 protein to another protein, e.g., a protein interacting with SPT4 (e.g., an SPT5 protein, such as Spt5 or SUPT5H), etc. In some instances, the subject compound diminishes interaction of the SPT4 protein and a second protein. In certain instances, the second protein is a SPT5 protein. The term SPT5 protein is used herein to collectively refer to not only yeast Spt5 proteins, but also mammalian homologs thereof, e.g., human SUPT5H; murine Supt5h, etc. In certain embodiments of the method, the subject compound diminishes interaction between Supt4h and Supt5h. Human Supt4h may form a DSIF complex with Supt5h, as may its yeast ortholog, to regulate transcription elongation (Guo et al., "Core structure of the yeast spt4-spt5 complex: a conserved module for regulation of transcription elongation," Structure (2008) 16: 1649-1658; Hatzog et al., " Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae," Genes Dev. (1998) 23:357-369; Wada et al., "DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs," Genes Dev (1998) 12: 343-356; Wenzel et al., "Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface," Biochem J (2009) 425: 373-380). In certain embodiments of the method, the compound diminishes interaction between RNA polymerase II and Supt5h or the DSIF complex. For example, a subject compound may interfere with binding of Supt 5h to RNA polymerase II, and its effects on the interaction between Supt4h and Supt5h may be indirect.
Also provided are methods of diminishing interaction of a SPT4 protein (e.g., as described herein) and a second protein in a sample by contacting the sample with an effective amount of a compound (e.g., as described herein) that differentially, if not selectively, diminishes the interaction of the SPT4 protein and the second protein. In certain instances, the second protein is a SPT5 protein (e.g., as described herein). By “diminishes interaction” is meant that the extent of binding of the SPT4 protein to the second protein (e.g., a fraction of bound SPT4 as compared to total SPT4) is reduced by 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or by 100% , e.g., as compared to a suitable control, e.g., a cell not contacted with the compound of interest. Any convenient methods may be utilized to determine extent of binding of the SPT4 protein to the second protein. In certain embodiments of the method, the compound diminishes interaction between Supt4h and Supt5h and disrupts the subsequent formation of the DSIF complex. The compound may specifically bind to the SPT4 protein and disrupt the interaction of the SPT4 protein with the SPT5 protein. In some instances, the compound specifically binds to the SPT5 protein and disrupts the interaction between the SPT4 and SPT5 protein. In some cases, by disrupting the interaction between the SPT4 and STP5 protein, the formation of the DSIF complex is also disrupted.
In some instances, an effective amount of a compound is an interaction diminishing amount, i.e. , an amount of the compound that inhibits the formation of a SPT4 complex (e.g., a SPT4/SPT5 (DSIF) complex) by 20% or more, such as 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more, as compared to SPT4 complex formation in the absence of the compound. Any convenient methods of assaying inhibition of complex formation or competitive inhibition may be utilized, such as those methods described by Cheng et al. “Selective reduction of the deleterious activity of extended tri-nucleotide repeat containing genes” WO 2012078906, the disclosure of which assay methods are herein incorporated by reference. Agents of interest include, but are not limited to, those described in Published PCT Application Publication Nos. WO2012078906; W02016196012; WO2018236910, and WO2020131573, the disclosures of which applications are herein incorporated by reference in their entirety.
In certain embodiments of the method, the subject compound modulates expression of extended repeat regions or domains that are not in genes (i.e. , that are in non-genic DNA), such as telomeric regions, e.g., telomeric regions that contain extended repeats, e.g., telomeric TTAGGG repeat containing regions. In some instances, the method reduces expression of such repeat regions, such as reducing expression of TERRA from telomeric repeat regions. In some embodiments, the method reduces transcription elongation, but not transcription initiation. In some cases, the method reduces the number of repeats transcribed in a repeat containing region such that the generated transcripts are relatively shorter and have fewer repeats than the transcripts from a control, e.g., the method reduces the number of repeats present in each TERRA transcript. In some instances, the method reduces the abundance of high molecular weight transcripts, e.g., high molecular weight TERRA transcripts. Any convenient assays may be used to determine a reduction in expression or a reduction in the number of repeats in the transcripts in a cell using the subject compounds relative to a control, e.g., a cell not contacted with the compound of interest, where the magnitude of expression reduction may be 10% or more, such as 20% or more, 30% or more, 50% or more, 100% or more, such as by 2-fold or more, by 5- fold or more, by 10-fold or more, by 20-fold or more, by 50-fold or more, by 100-fold or more, or even more. The magnitude of any difference in expression resulting from administration of the compound may vary, where in some instances the magnitude of reduction of expression relative to corresponding expression in a control is 2-fold or more, by 5- fold or more, by 10- fold or more, by 20-fold or more, by 50-fold or more, by 100-fold or more, or even more. In certain embodiments, the methods may reduce the deleterious impact of expression of a non-genic nucleotide repeat domain, such as telomeric regions that contain extended repeats, e.g., telomeric TTAGGG repeat containing regions.
In additional embodiments of the method, the subject compound reduces the ability of a cancer cell to use alternative lengthening of telomeres (ALT) pathways and break- induced replication (BIR) mechanisms to support telomere elongation and cell immortality. In some cases, the subject compound reduces cell proliferation and growth in cancer cells. In some embodiments, the subject compound suppresses or inhibits telomerase activity. Any convenient assays (e.g., as described herein) may be used to determine the effect of using the subject compounds relative to a control, e.g., a cell not contacted with the compound of interest, where the magnitude of reduction may be 10% or more, such as 20% or more, 30% or more, 50% or more, 100% or more, such as by 2-fold or more, by 5- fold or more, by 10-fold or more, by 20-fold or more, by 50-fold or more, by 100-fold or more, or even more. The magnitude of any differences resulting from administration of the compound may vary, where in some instances the magnitude of reduction relative to the corresponding effect in a control is 2-fold or more, by 5- fold or more, by 10-fold or more, by 20-fold or more, by 50-fold or more, by 100-fold or more, or even more.
In some instances, the subject compound increases apoptosis of cancer cells, e.g., ALT+ cancer cells. Any convenient assays (e.g., as described herein) may be used to determine the effect of using the subject compounds relative to a control, e.g., a cell not contacted with the compound of interest, where the magnitude of increase may be 10% or more, such as 20% or more, 30% or more, 50% or more, 100% or more, such as by 2-fold or more, by 5- fold or more, by 10-fold or more, by 20-fold or more, by 50-fold or more, by 100-fold or more, or even more. The magnitude of any differences resulting from administration of the compound may vary, where in some instances the magnitude of increase relative to the corresponding effect in a control is 2-fold or more, by 5- fold or more, by 10-fold or more, by 20-fold or more, by 50-fold or more, by 100-fold or more, or even more.
In practicing methods according to certain embodiments, an effective amount of an agent, e.g., such as described above, e.g., an SPT4 modulatory agent, is provided in the target cell or cells. In some instances, the effective amount of the compound is provided in the cell by contacting the cell with the compound. Contact of the cell with the modulatory agent may occur using any convenient protocol. The protocol may provide for in vitro or in vivo contact of the modulatory agent with the target cell, depending on the location of the target cell. In some instances, the cell is in vitro. In certain instances, the cell is in vivo. Contact may or may not include entry of the compound into the cell. For example, where the target cell is an isolated cell and the modulatory agent is an agent that modulates expression of SPT4, the modulatory agent may be introduced directly into the cell under cell culture conditions permissive of viability of the target cell. The choice of method is generally dependent on the type of cell being contacted and the nature of the compound, and the circumstances under which the transformation is taking place (e.g., in vitro, ex vivo, or in vivo).
Alternatively, where the target cell or cells are part of a multicellular organism, the modulatory agent may be administered to the organism or subject in a manner such that the compound is able to contact the target cell(s), e.g., via an in vivo or ex vivo protocol. By "in vivo,” it is meant in the target construct is administered to a living body of an animal. By “ex vivo” it is meant that cells or organs are modified outside of the body. Such cells or organs are in some cases returned to a living body.
In certain embodiments, the method is an in vivo method that includes: administering to a subject in need thereof an effective amount of an agent, e.g., as described above. The term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition in a patient, such as a mammal (such as a human) that includes: (a) preventing the disease or medical condition from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as, eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient.
In certain embodiments of the method, the disease or medical condition to be treated is a cellular proliferative disease. Cellular proliferative diseases are conditions characterized by abnormal cellular proliferation, such as cancers. Such disease conditions include cancer and neoplastic diseases and other diseases characterized by the presence of unwanted cellular proliferation, e.g., hyperplasias, and the like. In certain embodiments, the cellular proliferative disease to be treated is cancer.
To escape replicative senescence, cancer cells must overcome telomere attrition during DNA replication. Most cancer cells rely on telomerase to extend and maintain telomeres, but a significant fraction use the telomerase-independent pathway of alternative lengthening of telomeres (ALT) to overcome telomere shortening. In some cases, the cancer to be treated is a telomerase positive (TEL+) cancer. In some cases, the cancer to be treated is an alternative lengthening of telomeres positive (ALT+) cancer.
In some embodiments, the cancer to be treated is an ALT+ cancer. ALT+ cancers are characterized by the use of a homologous recombination-based ALT pathway to extend and maintain telomeres and acquire replicative immortality. In ALT+ cells, telomeres are elongated by break-induced replication (BIR), a repair process initiated by one-ended double-stranded breaks (DSBs) at collapsed replication forks and extended by conservative DNA replication. ALT is prevalent in cancers from the mesenchymal origin and usually associates with poor clinical outcome. ALT activity in cells and ALT+ cancers may be identified by the detection of one or more biomarkers, including, but not limited to, an absence of telomerase or telomerase activity, the presence of telomeric variant repeats (e.g., TAAGGG, TTAGAG, and TTTGGG); telomeres of substantial length (e.g., >50 kb); telomeres of heterogeneous length (e.g., <8 kb and >50 kb); elevated levels of telomere sister chromatid exchange (tSCE); extra-chromosomal telomeric repeats (ECTRs), particularly C-circles; ALT-associated acute promyelocytic leukemia (PML) bodies (APBs); and telomere dysfunction-induced foci (TIFs). Methods of detecting biomarkers are known in the art and widely used (see Zhang et al., "ALT Positivity in Human Cancers: Prevalence and Clinical Insights," Cancers (Basel) (2021) 13(10): 2384). Examples of cancers that may be ALT+ include, but are not limited to, osteosarcomas, breast cancers, gliomas, choroid plexus carcinomas, medullablastomas, neuroblastomas, pancreatic neuroendocrine tumors (PanNETs), angiosarcomas, leiomyosarcomas, lipsarcomas, and undifferentiated pleomorphic sarcomas (see Zhang et al. (2021 ), supra).
As used herein, the terms “host”, “subject”, “individual” and “patient” are used interchangeably and refer to any mammal in need of such treatment according to the disclosed methods. Such mammals include, e.g., humans, ovines, bovines, equines, porcines, canines, felines, non-human primate, mice, and rats. In certain embodiments, the subject is a non-human mammal. In some embodiments, the subject is a farm animal. In other embodiments, the subject is a pet. In some embodiments, the subject is mammalian. In certain instances, the subject is human. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees, and monkeys).
The amount of compound administered can be determined using any convenient methods to be an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms of the present disclosure will depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
In some embodiments, an effective amount of a subject compound is an amount that ranges from about 50 ng/ml to about 50 pg/ml (e.g., from about 50 ng/ml to about 40 pg/ml, from about 30 ng/ml to about 20 pg/ml, from about 50 ng/ml to about 10 pg/ml, from about 50 ng/ml to about 1 pg/ml, from about 50 ng/ml to about 800 ng/ml, from about 50 ng/ml to about 700 ng/ml, from about 50 ng/ml to about 600 ng/ml, from about 50 ng/ml to about 500 ng/ml, from about 50 ng/ml to about 400 ng/ml, from about 60 ng/ml to about 400 ng/ml, from about 70 ng/ml to about 300 ng/ml, from about 60 ng/ml to about 100 ng/ml, from about 65 ng/ml to about 85 ng/ml, from about 70 ng/ml to about 90 ng/ml, from about 200 ng/ml to about 900 ng/ml, from about 200 ng/ml to about 800 ng/ml, from about 200 ng/ml to about 700 ng/ml, from about 200 ng/ml to about 600 ng/ml, from about 200 ng/ml to about 500 ng/ml, from about 200 ng/ml to about 400 ng/ml, or from about 200 ng/ml to about 300 ng/ml). In some embodiments, an effective amount of a subject compound is an amount that ranges from about 10 pg to about 100 mg, e.g., from about 10 pg to about 50 pg, from about 50 pg to about 150 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 500 pg to about 750 pg, from about 750 pg to about 1 ng, from about 1 ng to about 10 ng, from about 10 ng to about 50 ng, from about 50 ng to about 150 ng, from about 150 ng to about 250 ng, from about 250 ng to about 500 ng, from about 500 ng to about 750 ng, from about 750 ng to about 1 pg, from about 1 pg to about 10 pg, from about 10 pg to about 50 pg, from about 50 pg to about 150 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 500 pg to about 750 pg, from about 750 pg to about 1 mg, from about 1 mg to about 50 mg, from about 1 mg to about 100 mg, or from about 50 mg to about 100 mg. The amount can be a single dose amount or can be a total daily amount. The total daily amount can range from 10 pg to 100 mg, or can range from 100 mg to about 500 mg, or can range from 500 mg to about 1000 mg.
In some embodiments, a single dose of the subject compound is administered. In other embodiments, multiple doses of the subject compound are administered. Where multiple doses are administered over a period of time, the DSIF complex modulating compound is administered twice daily (qid), daily (qd), every other day (qod), every third day, three times per week (tiw), or twice per week (biw) over a period of time. For example, a compound is administered qid, qd, qod, tiw, or biw over a period of from one day to about 2 years or more. For example, a compound is administered at any of the aforementioned frequencies for one week, two weeks, one month, two months, six months, one year, or two years, or more, depending on various factors.
Any of a variety of methods can be used to determine whether a treatment method is effective. For example, a biological sample obtained from an individual who has been treated with a subject method can be assayed for the presence and/or level of cells that are cancer cells, e.g., ALT+ cancer cells. Assessment of the effectiveness of the methods of treatment on the subject can include assessment of the subject before, during and/or after treatment, using any convenient methods. Aspects of the subject methods further include a step of assessing the therapeutic response of the subject to the treatment.
In some embodiments, the method includes assessing the condition of the subject, including diagnosing or assessing one or more symptoms of the subject which are associated with the disease or condition of interest being treated (e.g., as described herein). In some embodiments, the method includes obtaining a biological sample from the subject and assaying the sample, e.g., for the presence of a target gene or gene product or for the presence of cells that are associated with the disease or condition of interest (e.g., as described herein). The sample can be a cellular sample. In some cases, the sample is a biopsy. The assessment step(s) of the subject method can be performed at one or more times before, during and/or after administration of the subject compounds, using any convenient methods. In certain embodiments, the assessment step includes measuring the RNA or protein expression levels of SPT4. In some cases, the assessment step includes directly measuring the activity of SPT4, e.g., assaying for the association of SPT4 with SPT5 to form a DSIF complex or for the association of the DSIF complex with RNA pol II. In some embodiments, the assessment step includes indirectly measuring the activity of SPT4, e.g., assaying for the presence or abundance of the DSIF complex, assaying for TERRA expression, assaying for the presence of ALT+ biomarkers, assaying for telomere length, or assaying for telomerase activity as described herein. In certain cases, the assessment step includes identification of cells including cancer cells. In some embodiments, the cancer cells are ALT+ cancer cells. In other embodiments, the cancer cells are TEL+ cells. In some cases, identification of cells includes determining whether the cells are apoptotic or viable or assaying for cell growth or proliferation. In certain instances, assessing the subject includes diagnosing whether the subject has a disease or condition of interest. In some instances, assessing the subject includes determining whether the subject has a symptom associated with the disease or condition of interest.
In some instances, the method delays occurrence of a symptom associated with the disease. In certain instances, the method reduces the magnitude of a symptom associated with the disease.
The term "surrogate marker" is employed in its conventional sense to refer to a measure of the effects of specific disease treatment or predict outcomes in a clinical trial. Surrogate markers can be defined as a laboratory measurement or a physical sign that is used in therapeutic trials as a substitute for a clinically meaningful endpoint. Reliable surrogates, rigorously validated in phase III clinical trials, can forecast the long term effect of the therapy based on how the patient feels, functions, or survives (Katz, “Biomarkers and Surrogate Markers: an FDA Perspective,” NeuroRx (2004) 1 : 189-95). These markers may also be used to compare drug efficacy between trials and may even become the basis for which new drugs gain regulatory approval for marketing (Twaddell, “Surrogate outcome markers in research and clinical practice,” Australian Prescriber (2009) 32: 47-50). Because their use can reduce the size, duration, and cost of large studies or clinical trials, these markers are especially valuable if the predicted drug effect prevents death or promotes other critically important outcomes. For some progressive diseases, surrogate markers may be able to determine the disease stage (Weston, “The use of surrogate end points in cardiovascular disease and diabetes,” The British Journal of Cardiology (2008) 15: S6-S7). Depending on the specific disease condition, surrogate markers may vary widely. Embodiments of the present disclosure therefore include administering a compound, e.g., as described herein, to modulate, e.g., improve, one or more surrogate markers of the disease condition.
In the subject methods, the compound (e.g., as described herein) may be administered to the targeted cells using any convenient administration protocol capable of resulting in the desired activity. Thus, the subject compounds can be incorporated into a variety of formulations, e.g., pharmaceutically acceptable vehicles, for therapeutic administration. As reviewed above, the subject methods result in reduction in the deleterious activity of an extended target repeat in a target cell or cells, where the target cell(s) may be in vitro or in vivo. In certain embodiments, the subject methods result in reduction in toxicity of a target gene, e.g., via a reduction in aggregation of a protein encoded thereby, in a target cell(s) . In certain embodiments, the methods result in enhancement in function of a protein encoded by a target gene.
PHARMACEUTICAL PREPARATIONS
Also provided are pharmaceutical preparations. Pharmaceutical preparations are compositions that include an active agent (e.g., as described herein) (for example one or more of the subject compounds, either alone or in the presence of one or more additional active agents) present in a pharmaceutically acceptable vehicle. "Pharmaceutically acceptable vehicles" may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which a compound of the present disclosure is formulated for administration to a mammal. Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
When administered to a mammal, the compounds and compositions of the present disclosure and pharmaceutically acceptable vehicles, excipients, or diluents may be sterile. In some instances, an aqueous medium is employed as a vehicle when the subject compound is administered intravenously, such as water, saline solutions, and aqueous dextrose and glycerol solutions.
Pharmaceutical compositions can take the form of capsules, tablets, pills, pellets, lozenges, powders, granules, syrups, elixirs, solutions, suspensions, emulsions, suppositories, or sustained-release formulations thereof, or any other form suitable for administration to a mammal. In some instances, the pharmaceutical compositions are formulated for administration in accordance with routine procedures as a pharmaceutical composition adapted for oral or intravenous administration to humans. Examples of suitable pharmaceutical vehicles and methods for formulation thereof are described in Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, Chapters 86, 87, 88, 91 , and 92, incorporated herein by reference. The choice of excipient will be determined in part by the particular compound, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the subject pharmaceutical compositions.
Administration of the subject compounds may be systemic or local. In certain embodiments administration to a mammal will result in systemic release of a compound of the present disclosure (for example, into the bloodstream). Methods of administration may include enteral routes, such as oral, buccal, sublingual, and rectal; topical administration, such as transdermal and intradermal; and parenteral administration. Suitable parenteral routes include injection via a hypodermic needle or catheter, for example, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intraarterial, intraventricular, intrathecal, and intracameral injection and non-injection routes, such as intravaginal rectal, or nasal administration. In certain embodiments, the compounds and compositions of the present disclosure are administered subcutaneously. In certain embodiments, the compounds and compositions of the present disclosure are administered orally. In certain embodiments, it may be desirable to administer one or more compounds of the present disclosure locally to the area in need of treatment. This may be achieved, for example, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
The compounds can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
A subject compound may also be formulated for oral administration. For an oral pharmaceutical formulation, suitable excipients include pharmaceutical grades of carriers such as mannitol, lactose, glucose, sucrose, starch, cellulose, gelatin, magnesium stearate, sodium saccharine, and/or magnesium carbonate. For use in oral liquid formulations, the composition may be prepared as a solution, suspension, emulsion, or syrup, being supplied either in solid or liquid form suitable for hydration in an aqueous carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, preferably water or normal saline. If desired, the composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers. In some embodiments, formulations suitable for oral administration can include (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, or saline; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can include the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles including the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are described herein.
The subject formulations can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They may also be formulated as pharmaceuticals for non-pressured preparations such as for use in a nebulizer or an atomizer.
In some embodiments, formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
Formulations suitable for topical administration may be presented as creams, gels, pastes, or foams, containing, in addition to the active ingredient, such carriers as are appropriate. In some embodiments the topical formulation contains one or more components selected from a structuring agent, a thickener or gelling agent, and an emollient or lubricant. Frequently employed structuring agents include long chain alcohols, such as stearyl alcohol, and glyceryl ethers or esters and oligo(ethylene oxide) ethers or esters thereof. Thickeners and gelling agents include, for example, polymers of acrylic or methacrylic acid and esters thereof, polyacrylamides, and naturally occurring thickeners such as agar, carrageenan, gelatin, and guar gum. Examples of emollients include triglyceride esters, fatty acid esters and amides, waxes such as beeswax, spermaceti, or carnauba wax, phospholipids such as lecithin, and sterols and fatty acid esters thereof. The topical formulations may further include other components, e.g., astringents, fragrances, pigments, skin penetration enhancing agents, sunscreens (e.g., sunblocking agents), etc.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet, or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may include the inhibitor(s) in a composition as a solution in sterile water, normal saline, or another pharmaceutically acceptable carrier.
The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present disclosure calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the novel unit dosage forms of the present disclosure depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host. In pharmaceutical dosage forms, the compounds may be administered in the form of a free base, their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
Dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Desired dosages for a given compound are readily determinable by a variety of means. The dose administered to an animal, particularly a human, in the context of the present disclosure should be sufficient to effect a prophylactic or therapeutic response in the animal over a reasonable time frame, e.g., as described in greater detail herein. Dosage will depend on a variety of factors including the strength of the particular compound employed, the condition of the animal, and the body weight of the animal, as well as the severity of the illness and the stage of the disease. The size of the dose will also be determined by the existence, nature, and extent of any adverse sideeffects that might accompany the administration of a particular compound. COMBINATION T HERAPY
In some instances, the DSIF complex modulating agents may be used jointly with any agent useful in the treatment of a neoplastic condition, such as anti-cancer agents and anti-tumor agents. Agents of interest which can be used jointly with the subject DSIF modulating compounds in such instances include, but are not limited to, Cancer chemotherapeutic agents, Agents that act to reduce cellular proliferation, Antimetabolite agents, Microtubule affecting agents, Hormone modulators and steroids, natural products, and biological response modifiers, e.g., as described in greater detail below.
Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinaceous) compounds that reduce proliferation of cancer cells and encompass cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds can also be used. Suitable cancer chemotherapeutic agents include dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See, e.g., WO 96/33212, WO 96/14856, and U.S. 6,323,315. For example, dolastatin 10 or auristatin PE can be included in an antibody-drug conjugate of the present disclosure. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1 -TM1); and benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepine (PBD). Agents that act to reduce cellular proliferation are known in the art and widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide. Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatine, and gemcitabine. Suitable natural products and their derivatives, (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins), include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.; antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like. Other anti-proliferative cytotoxic agents are navelbene, CPT-11 , anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine. Microtubule affecting agents that have antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, epothilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.
Hormone modulators and steroids (including synthetic analogs) that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g. aminoglutethimide; 17a-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®. Estrogens stimulate proliferation and differentiation. Therefore, compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids may inhibit T cell proliferation. Other suitable chemotherapeutic agents include metal complexes, e.g., cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g., hydroxyurea; and hydrazines, e.g., N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other anti-proliferative agents of interest include immunosuppressants, e.g., mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4- morpholinyl)propoxy)quinazoline) ; etc.
Taxanes are suitable for use. “Taxanes” include paclitaxel, as well as any active taxane derivative or pro-drug. “Paclitaxel” (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3’N- desbenzoyl-3’N-t-butoxycarbonyl analogs of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881 , WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtained from a variety of commercial sources, including for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912 from Taxus yannanensis). Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., Taxotere™ docetaxel, as noted above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose). Also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazino and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021 , WO 98/22451 , and U.S. Patent No. 5,869,680; 6- thio derivatives described in WO 98/28288; sulfenamide derivatives described in U.S. Patent No. 5,821 ,263; and taxol derivative described in U.S. Patent No. 5,415,869. It further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98/58927; WO 98/13059; and U.S. Patent No. 5,824,701. Biological response modifiers suitable for use include, but are not limited to, (1 ) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine/threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-a; (7) IFN-y; (8) colony-stimulating factors; and (9) inhibitors of angiogenesis.
In some instances, the agents of the invention are employed in combination with immunotherapy agents. Examples of immunotherapy include anti-PD-1/PD-L1 immunotherapies, such as anti-PD-1/PD-L1 therapeutic antagonists, where such antagonists include but are not limited to e.g., OPDIVO® (nivolumab), KEYTRUDA® (pembrolizumab), Tecentriq™ (atezolizumab), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS-936559 (MDX-1105), CA-170, BMS-202, BMS-8, BMS-37, BMS-242 and the like. Nivolumab (OPDIVO®) is a humanized lgG4 anti-PD-1 monoclonal antibody used to treat cancer. Pembrolizumab (KEYTRUDA®), formerly known as MK-3475, lambrolizumab, etc., is a humanized antibody used in cancer immunotherapy targeting the PD-1 receptor. Atezolizumab (Tecentriq™) is a fully humanized, engineered monoclonal antibody of lgG1 isotype against the PD-L1 protein. Durvalumab (Medlmmune) is a therapeutic monoclonal antibody that targets PD-L1 . Avelumab (also known as MSB0010718C; Merck KGaA, Darmstadt, Germany & Pfizer) is a fully human monoclonal PD-L1 antibody of isotype lgG1 . BMS-936559 (also known as MDX-1105; Bristol-Myers Squibb) is a blocking antibody that has been shown to bind to PD-L1 and prevent its binding to PD-1 (see e.g., U.S. NIH Clinical Trial No. NCT00729664). CA-170 (Curis, Inc.) is a small molecule PD-L1 antagonist. BMS-202, BMS-8, BMS-37, BMS-242 are small molecule PD- 1/PD-L1 complex antagonists that bind PD-1 (see e.g., Kaz et al., (2016) Oncotarget 7(21); the disclosure of which is incorporated herein by reference in its entirety). Anti-PD-L1 antagonists, including e.g., antibodies, useful in the methods described herein include but are not limited to e.g., those described in U.S. Patent Nos. 7,722,868; 7,794,710;
7,892,540; 7,943,743; 8,168,179; 8,217,149; 8,354,509; 8,383,796; 8,460,927; 8,552,154; 8,741 ,295; 8,747,833; 8,779,108; 8,952,136; 8,981 ,063; 9,045,545; 9,102,725; 9,109,034; 9,175,082; 9,212,224; 9,273,135 and 9,402,888; the disclosures of which are incorporated herein by reference in their entirety. Anti-PD-1 antagonists, including e.g., antibodies, useful in the methods described herein include but are not limited to e.g., those described in 6,808,710; 7,029,674; 7,101 ,550; 7,488,802; 7,521 ,051 ; 8,008,449; 8,088,905; 8,168,757; 8,460,886; 8,709,416; 8,951 ,518; 8,952,136; 8,993,731 ; 9,067,998; 9,084,776; 9,102,725; 9,102,727; 9,102,728; 9,109,034; 9,181,342; 9,205,148; 9,217,034; 9,220,776; 9,308,253; 9,358,289; 9,387,247 and 9,402,899; the disclosures of which are incorporated herein by reference in their entirety.
The terms "co-administration" and "in combination with" include the administration of two or more therapeutic agents either simultaneously, concurrently, or sequentially within no specific time limits. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.
"Concomitant administration" of a known therapeutic drug with a pharmaceutical composition of the present disclosure means administration of the compound and second agent at such time that both the known drug and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a subject compound. Routes of administration of the two agents may vary, where representative routes of administration are described in greater detail below. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compounds of the present disclosure.
In some embodiments, the compounds (e.g., a subject compound and the at least one additional compound) are administered to the subject within twenty-four hours of each other, such as within 12 hours of each other, within 6 hours of each other, within 3 hours of each other, or within 1 hour of each other. In certain embodiments, the compounds are administered within 1 hour of each other. In certain embodiments, the compounds are administered substantially simultaneously. By administered substantially simultaneously is meant that the compounds are administered to the subject within about 10 minutes or less of each other, such as 5 minutes or less, or 1 minute or less of each other.
UTILITY
The subject methods and compound compositions find use in treatment of a subject for a disease condition, such as a cellular proliferative disease condition, e.g., a cancer, such as an ALT+ cancer. By treatment is meant at least an amelioration of one or more symptoms associated with the disease condition afflicting the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the pathological condition being treated, such as loss of cognitive function, etc. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition. Treatment may also manifest in the form of a modulation of a surrogate marker of the disease condition, e.g., as described above.
A variety of hosts are treatable according to the subject methods. Generally such hosts are "mammals" or "mammalian," where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs and rats), and primates (e.g., humans, chimpanzees and monkeys). In some embodiments, the host is human.
The subject methods and compound compositions also find use in modulating transcription mediated by the DSIF complex comprising SPT4 and SPT5. In particular, the subject methods and compound compositions also find use in modulating transcription elongation, reducing the transcription of RNA with extended repeat regions (e.g., TERRA, pathogenic RNA in HD or ALS, etc.) or the number of repeats transcribed, and research in transcription, ALT mechanisms, the role of TERRA, and telomere lengthening. Furthermore, the subject methods and compound compositions find use in the characterization of ALT+ and ALT- cancers. The subject methods and compound composition also find use in modulating telomerase dysfunction and treating associated cancers thereof.
KITS & SYSTEMS
Also provided are kits and systems that find use in practicing embodiments of the methods, such as those described as described above. The term "system" as employed herein refers to a collection of two or more different active agents, present in a single or disparate composition, that are brought together for the purpose of practicing the subject methods. The term kit refers to a packaged active agent or agents. In some embodiments, the subject system or kit includes a dose of a subject compound (e.g., as described herein) and a dose of a second active agent (e.g., as described herein) in amounts effective to treat a subject for a disease or condition associated with the deleterious activity of an extended nucleotide repeat containing target region. Kits and systems for practicing the subject methods may include one or more pharmaceutical formulations. As such, in certain embodiments the kits may include a single pharmaceutical composition, present as one or more unit dosages, where the composition may include one or more nucleoside compounds (e.g., as described herein). In some embodiments, the kit may include two or more separate pharmaceutical compositions, each containing a different active agent, at least one of which is a nucleoside compound (e.g., as described herein).
Also of interest are kits and systems finding use in the subject methods, e.g., as described above. Such kits and systems may include one or more components of the subject methods, e.g., nucleoside agents, cells, vectors encoding proteins of interest, enzyme substrates, dyes, buffers, etc. The various kit components may be present in the containers, e.g., sterile containers, where the components may be present in the same or
Figure imgf000031_0001
In addition to the above-mentioned components, subject kits may further include instructions for using the components of the kit, e.g., to practice the subject method. The instructions are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e. , associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, Hard Disk Drive (HDD), portable flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
The following examples are offered by way of illustration and not by way of limitation.
EXAMPLES
I. Introduction
Spt4/Spt5 is a highly conserved transcription elongation complex which regulates polymerase II processivity. The human orthologue Supt4 is selectively required for the expression of long trinucleotide repeat containing transcripts in multiple neurodegenerative diseases models. In this manuscript, the function of Supt4 is further investigated for the transcription of the non-coding RNA TERRA, the (TTAGGG)6 repeat sequence transcribed from the chromosome telomere region. We have discovered that supt4 depletion decreases TERRA repeat abundance in U2OS, an osteosarcoma cell line which harbors extremely long TERRA due to the mechanism of Alternative Lengthening of Telomere, and consequently disrupts telomere recombination, shortens telomere and causes apoptosis.
II. MATERIALS AND METHODS
A. Cell culture
Three human cancer cell lines, Hela, MG-63 and U2OS, which were routinely cultured in 5% CO2 at 37°C with DMEM+10% FBS. Human fibroblast cell lines BJ and GM8747 (ICF patient) were cultured with DMEM+15% FBS. Human lymphocytes were cultured with RPM1160+10% FBS. B. siRNA Knock-down and Ienti-supt4 knock-down siRNA transfection was performed with various amounts of supt4 siRNA and Ctrl RNA pool from Damacon. Thermofisher lipofectamine RNAimax regent was used for the transfection. Cells were collected after various time. For 240 h siRNA knock-down, cells were collected after 5 days of siRNA treatment and reseeded, second transfection was performed 12 hours after seeding.
The supt4 shRNA lenti-virus was added to the cell culture together with polybrene (8 pg/ml), and 1 pg/ml puromycin was used for selection.
C. DNA extraction, and TRF analysis to determine telomere length
Genomic DNA was extracted using DNeasy kit (Qiagen) according to manufacturer’s protocol. For TRF length measurement, 10pg of high molecular weight genomic DNA were digested with the Rsal and Hif I restriction enzymes (NEB biolabs). After electrophoresis in 0.6% agarose gel, DNA was denatured and transferred to nylon membrane (Amersham Hybond-N+, GE healthcare). The hybridization procedure was performed according to the protocol (Zhou S, 2011]. Briefly, The membranes were hybridized in 0.5M phosphate buffer with 7% SDS and 1 mM EDTA containing 32P-y[ATP]-labeled probe generated by endlabeling of (TTAGGG)4 oligo for 18 hours at 50°C . After hybridization, the membranes were washed in 4XSSC 0.1%SDS twice at room temperature, then in 2XSSC 0.1%SDS twice at 50°C. Radioactive signal were detected using a phosphoimager.
D. Quantification of C circle formation
Quantification of C circle formation in U2OS cells was performed according the protocol published [Henson JD, 2009]. Briefly, 20 pl reaction was assembled and incubated at 30°C for 8 hours with 1/3 serial diluted genomic DNA starting from 432ng DNA in 6 ul H2O. (10 ug/ul BSA: 0.4 pl; 20% tween-20: 0.1 pl; 0.1 M DTT: 0.8 pl; 10Xbuffer: 2 ul; 10mMdATP, dGTP, dTTP mix: 2.5 pl; Phi 29: 0.7 pl). The reaction was diluted with 40 pl 2XSSC, slot blot to hybound-N positively charged nylon (Amersham). The hybridization was performed using the same protocol as telomere TRF assay using 32P-y[ATP]-labeled probe generated by end-labeling of (AATCCC)4 oligo.
E. RNA extraction, high MW Northern and dot blotting
RNA from cancer cells was extracted using RNeasy kit with on column DNase I digestion (Qiagen), and RNA from fibroblast was extracted with Trizol regent first (Thermo fisher), then purified with RNeasy kit with on column DNase I digestion. High MW Northern was performed as described. 10 ug RNA samples were treated in 30 ul denaturing solution for 2 min at 80°C (deionized formamide: 13.125 ul; 40% formaldehyde: 3.75 ul; 10X MOPS buffer: 3 ul; 2 mg/ml EtBr: 0.375 ul; Formamide loading dye: 1 ul), then put on ice. The denatured RNA samples were run in agarose gel containing 7% formaldehyde with 1XMOPS buffer containing 0.7% formaldehyde. 100V for 1 h, then 250V 2h, +/- buffer was changed every 30 min. Gel was washed 3 times with DEPC-treated water, then treated in gel denaturing buffer containing 50mM NaOH and1 .5 mM NaCI for 30min. After washed by 20X SSC for 45 min, gel was semi-dry transferred to nylon membrane in 10XSSC O.N. Membrane was rinsed in 2XSSC, crosslinked by 125 mJ UV, then baked at 80 °C for 1 hour. Hybridization was performed in Ultrahyb-oligo buffer with 32P-Y[ATP]-labeled (AATCCC)4 oligo at 42°C O.N. Membrane was washed by 2X SSC 0.1%SDS RT for 10 min, then 2X SSC 0.5%SDS at 42V for 30 min. For dot-blotting, 5 pg RNA was denatured by adding equal volume of Northern-Max Gly sample loading dye (Invitrogen) at 50°C for 1 hour, then slot blot to Hybound-N+. Ultrahyb-oligo buffer with 32P-y[ATP]-labeled (AATCCC)4 oligo was used for hybridization at 42°C for 48 hours. Membranes were washed twice with 2XSSC 0.1%SDS and exposed to phosphor image. The radioactive signal was detected by phosphor image.
F. Real time PCR quantification of TERRA
5 ug RNA was used for reverse transcription using superscript III (Thermo Fisher) with (AATCCC)e and GAPDH primer. Chromosome-specific TERRA quantification was performed following the published protocol on Thermo Fisher QS3 [Feretzaki M, 2017] .
G. Cell viability and toxicity
1500 cells were seeded per well, in triplicate, of a 96-well plate and incubated overnight. The following day cells were either left untreated, or treated with supt4 siRNA or control siRNA. The cells were incubated for 4 days and cell viability was analyzed using CellTiter Gio and Tecan plate reader. For analysis of cell death, cells were seeded in a 6- well dish at 0.5 x 105 and allowed to incubate for 8 hr. Cells were either left untreated, or treated with supt4 siRNA or control siRNA for 6 or 12 days. Cells were collected by trypsin and stained for FACS analysis using the Annexin V assay kit (Life Technologies) per the manufacturer’s recommendations. Cell death was analyzed using Flowjo software.
III. RESULTS
A. Supt4 Knock-down decreases long TERRA repeat abundance in U2OS cells TERRA transcripts displayed big length heterogeneity [Azzalin CM, 2007], The first correlation between TERRA length and telomere length was reported in fibroblasts derived from patients with ICF syndrome (Immunodeficiency, Centromeric region instability, Facial anomalies). The elongation of telomeres caused by overexpression of telomerase is accompanied with increase in the length of TERRA transcripts [Yehezkel S, 2008]. It was further proved that TERRA length is dependent on telomere length using various human cell lines with over-elongated telomere caused by ectopic telomerase overexpression [Arnoult N, 2012; Van Beneden A, 2013],
It is ideal to use a system with abundant and elongated TERRA to test the function of elongation factor Supt4 on the TERRA repeat transcription. We decided to use U2OS cell from ALT+ osteosarcoma. To maintain intact chromosome and to avoid replicative senescence, most human cancers reactivate the reverse transcriptase telomerase [Artandi, S.E., 2010]. However, a significant proportion (~5-15%) utilize a homologous recombination (HR) based telomere maintenance mechanism known as alternative lengthening of telomeres (ALT) [Cesare, A. J., 2010]. ALT cells have highly heterogeneous, fluctuating telomere lengths [Bryan, T.M., 1995], and high levels of telomere sister chromatid exchanges (t-SCEs) [Londono-Vallejo, J. A., 2004], abundant extrachromosomal telomeric repeat DNA (ECTR) [Cesare, A. J, 2004; Nabetani, A., 2009]. One major character of ALT+ cancer cells is elevation of TERRA, due to increased transcription and loss of its cell cycle regulation because of lack of ATRX activity [Clynes, D., 2015].
Various concentrations of supt4 siRNA were used to knock down Supt4 abundance in U2OS cells for 5 days and 10 days respectively (FIG. 8), then dot-blotting was used to measure the UUAGGG repeat abundance of TERRA transcript in the RNA sample. As shown in FIG. 1A and quantified in FIG. 1 B, UUAGGG repeat abundance in all the U2OS cells treated with supt4 siRNA was decreased up to 90% compared with the pooled siRNA control (FIG. 1 A & 1 B, supt4 KN vs siRNA Ctrl). The samples of U2OS cells treated with 20 nM supt4 siRNA and control siRNA were also tested for the transcript abundance of chromosome specific TERRA RNA using real-time PGR analysis with primer sets from 6 different subtelomeric regions (chromosome 1 , 2, 9, 10, 13, 15) (FIG. 1 C). There is no significant down-regulation of TERRA subtelomeric transcript abundance detected in the supt4 KN sample accompanied with the decrease of UUAGGG repeat abundance observed in FIG. 1 A & 1 B. Since CpG-island promoters which drive TERRA transcription is in the subtelomeric regions of chromosomes [Nergadze, S., 2009], real-time data in FIG. 1 C proves the molecular number of TERRA transcripts is not affected by supt4 depletion, indicating Supt4 doesn’t regulate TERRA transcription initiation. Therefore, the observed decrease of UUAGGG repeat abundance of TERRA (FIG. 1 A & 1 B) means shorter transcripts with fewer repeats, indicating transcription elongation is suppressed by supt4 depletion.
To further prove this notion, high MW Northern-blotting was performed with the RNA extracted from supt4 siRNA treated U2OS cells compared with pool siRNA treated cells. The results in FIG. 2 clearly show that knock-down of supt4 in U2OS cells significantly decreases TERRA transcript abundance in the high MW region, indicating Supt4 is required for the transcription elongation of extremely long TERRA RNA.
B. Supt4 KN inhibits Alternative Lengthening of Telomere (ALT) in U2OS cells
Emerging evidence shows TERRA are essential for the proliferation of ALT+ cancer cells, since TERRA enables DNA-RNA hybrids “R loop” formation at chromosome ends which can promote homologous recombination among telomeres to sustain genome instability and avoid cellular senescence [Arora, R., 2014], The fact that supt4 KN decreases long TERRA repeat abundance indicates the depletion of supt4 function may be connected to inhibition of alternative lengthening of telomere (ALT) in U2OS cells.
Partially single-stranded telomeric (CCCTAA)n circle (C-circles) generated by telomere recombination are specific and quantifiable markers of ALT [Grudic, A., 2007], C circle assay (CO assay) has been widely used to distinguish ALT tumors, which is the rolling circle amplification of partially double stranded C-circles by 29 DNA polymerase which is auto-primed by the partial G-strand ([TTAGGG]n), producing long telemetric ssDNA concatemers which can be detected by dot-blotting [Henson, J., 2009]. The result of CC assay in FIG. 3A shows Ienti-supt4 knock-down (FIG. 3B & 3C) causes inhibition of C circle amplification (FIG. 3A) accompanied with decrease of telomere TTAGGG repeat abundance (FIG. 3D) detected by real-time PCR analysis with 36B4 as the single copy gene control [method developed by O'Callaghan NJ, 2011]. Moreover, the quantification of southern-TRF data in FIG. 4 clearly show that long telomere signal in high molecular weight region is significantly decreased by Ienti-supt4 knock-down, whereas there is no obvious telomere signal intensity change in the low MW region (FIG. 4), proving supt4 knock-down causes telomere shortening in U2OS cells.
Inhibition of U2OS telomere recombination caused by supt4 depletion was not observed in the cells with 72 hours supt4 siRNA treatment (FIG. 9), indicating certain generations of cell culture is required for the effect to be visualized.
C. Supt4 KN inhibits the proliferation of both ALT' and ALT+ cancer cells.
The cell numbers of supt4 siRNA treated U2OS was counted after 5 days and 10 days of incubation (FIG. 4A, FIG. 9), and dose-dependent decrease was detected. Moreover, 10 days supt4 siRNA treatment causes more significant effect compared with 5 days treatment. It is consistent with the data in FIG. 1 -3 that supt4 knock down inhibits the ALT in U2OS cells, which is essential for the cell proliferation.
Hela is an immortal cervical cancer cell line which have an active telomerase to maintain the rapid cell growth [Ivankovic, M., 2007], and there is no observed ALT in Hela cell, therefore, Hela is considered as ALT". Similar growth inhibitory effect was observed in Hela cells (FIG. 4B), indicating there is more targets of supt4 function in cancer cells.
D. Supt4 knock-down increased the ratio of apoptotic cells in U2OS (ALP), but not in Hela (ALT )
Since maintain telomere is essential for cancer cell survival, FACS was used to determine whether supt4 depletion in U2OS can lead to apoptosis in this ALT+ cancer cells. The immortal cervical cancer cell line Hela (ALT-) was used as the negative control, since it harbors an active telomerase to maintain the rapid cell growth [Ivankovic, M., 2007], After being treated with 10nM supt4 siRNA for 10 days, the cells were stained with two dyes: Annexin V stains apoptotic cells by its ability to bind to phosphatidylserine, and PI stains dead cells. Recombinant Annexin V is conjugated to Alexa Fluor 488, which is a perfect spectral match to fluorescein (FITC). As shown in FIG. 5, Q1 (PI-, FITC+) are apoptotic cells with intact cell membrane and elevated phosphatidylserine; Q2 (PI+, FITC+) are late apoptotic cells with damaged cell membrane and elevated phosphatidylserine; Q3 (PI+, FITC-) represent necrotic cells; Q4 (PI-, FITC) are viable cells.
Between U2OS (ALT+) and Hela (ALT ), FACS data show the difference of the ratio of apoptotic cells caused by supt4 knock down (FIG. 5). Supt4 siRNA treatment caused significant increase of late apoptotic (Q2) cells and decrease of viable cells (Q4) in U2OS (FIG. 5A), which was not observed in Hela cells (FIG. 5B). We speculate that Supt4 knock down affects the proliferation of U2OS (ALT+) by inducing apoptosis triggered by ALT inhibition, whereas Supt4 knock down supt4 only delays cell growth of Hela (ALT-) without affecting apoptosis.
E. Supt4 shows no effect on TERRA repeat abundance in SAOS-2 and MG-63 cells
Furthermore, we tested the effect of supt4 KN in two other commonly used osteosarcoma cells SAOS-2 and MG-63 (FIG. 10). Similar procedure of TERRA UUAGGG repeat detection by dot-blotting was performed with samples with supt4 depletion to deplete cellular supt4 (FIG. 6A). Surprisingly, TERRA repeat abundance was not affected by supt4 depletion in SAOS and MG-63 after 10 day treatment (FIG. 6), neither was C circle formation (FIG. 11). Moreover, the HD143 compound disrupting Supt4 function also shows different growth inhibitory effect in U2OS and MG-63 cells (FIG. 12)
There is no difference regarding cellular level of supt4 (FIG. 10). One major difference of these three ALT+ cells are the proficiency of cancer suppressor P53. U2OS has functional P53, whereas SAGS and MG-63 are P53 deficient [Montanaro L, 2007], Higher TERRA repeat abundance in U2OS cells (FIG. 6) could be one of the evidences of P53 function. Recent publications show that P53 could be co-immunoprecipitated with active forms of DNA-directed RNA polymerase II subunit 1 (RPB1 ), highlighting its association with the elongating RNA polymerase II. [Borsos, 2017], Earlier work using high- resolution chromatin immunoprecipitation assays (ChIP) demonstrate that P53 can recruit multiple elongation factors including DSIF (DRB Sensitivity-Inducing Factor, Spt4/5 complex), P-TEFb (Positive Transcription Elongation Factor b), TFIIH, TFIIF, and FACT (Facilitates Chromatin Transcription) to distinct regions of the p21 (CIP1 ) locus, concomitantly with changes in RNAP II phosphorylation status [Gomes NP, 2006]. Moreover, P53-dependent regulation of transcriptional elongation regulator TFHS.h was reported to selectively promote transcription elongation of bax gene, an apoptosis- associated target of p53 in mammalian cells [Liao JM, 2016]. Further experiment are being performed to clarify the function of P53 in Supt4 regulation in ALT+ cells.
F. Supt4 knock down inhibits telomerase activity in ALT cancer cells
Hela is an immortal cervical cancer cell line which relies on active telomerase to maintain the rapid cell growth [Ivankovic, M., 2007], It is possible that Supt4 knock down suppresses telomerase activity to cause growth delay. Telomerase is a ribonucleoprotein responsible for maintaining telomere length. The core of telomerase has two components: Catalytic telomerase reverse transcriptase (TERT) and telomerase RNA (TERC). TERT utilizes the template region (3’-CAAUCCCAAUC-5’) of TERC to add TTAGGG DNA repeats and thereby extend single stranded 3' telomeric strands [Morin, G.B., 1989]. Telomerase have several accessory proteins including telomerase cajal body protein 1 (TCAB1 ), the four H/ACA-motif RNA binding proteins dyskerin, NHP2, NOP10, Gar1 , and the two ATPase proteins Pontin and Reptin [Xu, Y„ 2016].
The telomerase activity of Hela was tested by TRAP assay combined with real-time analysis (TRAPeze-RT), as well as telomerase activity of ALT U2OS and normal fibroblast BJ (FIG. 7A). The result shows Hela harbors very strong telomerase activity compared with the normal fibroblasts which has no active telomerase (FIG. 7A). And Supt4 knock down suppresses telomerase activity in Hela to 50% (FIG. 7B), which is consistent with the inhibition of its proliferation caused by supt4 Knock-down (FIG. 13), TERRA gets transcribed from the telomeric C-rich strand and therefore harbors sequences that are complementary to the RNA subunit (template region) of telomerase. As expected, TERRA acts as a potent in vitro inhibitor of telomerase, in part, by directly base pairing to the telomerase RNA moiety [Redon, S., 2010]. However, recent publications suggest that in vivo TERRA may positively affect telomerase function. TERRA might assist telomere lengthening in telomerase positive cells by recruiting telomerase to the shortest telomeres in cells [Moravec M, 2016]. Depleting TERRA levels in cancers could simultaneously suppress telomerase-mediated and ALT-mediated telomere elongation and therefore progression of different cancer types. [Cusanelli E, 2013]. And interestingly, Alternative Lengthening of Telomeres can coexist with active Telomerase [Perrem, K., 2001],
IV. DISCUSSION
In the previous publications, transcriptional Supt4 is required to facilitate the transcription of the pathogenic RNA with abnormally long trinucleotide repeats in neurodegenerative disease HD and ALS [Kramer NJ, 2016; Cheng HM, 2015; Liu CR, 2012], In this manuscript, Supt4 function was tested in osteosarcoma ALT+ cancer cells (FIG. 1-3), ICF fibroblast (FIG. 5A&B), normal fibroblast (FIG. 5C), and lymphocytes from neurodegenerative diseases (FIG. 6). Supt4 depletion was found to decrease TERRA repeat abundance in U2OS cells (FIG. 1 ), but to increase TERRA transcription in fibroblasts and lymphocytes (FIG. 5&6). The speculated reason for cell-specific supt4 regulation is the TERRA length variation caused by telomere variation in different cell types.
There are a lot of publications and discussions regarding TERRA transcription, regulation, and function in the recent years. However, the majority TERRA mechanisms still remains unknown. One of the reasons might be the technical challenges of TERRA detection - long RNA of heterogeneous sizes [Beneden AV, 2013, Diman A, 2018]. There are three molecular biology methods to detect TERRA: Northern-blotting, dot-blotting, and real-time PGR. Each method has limitations. Theoretically Northern-blotting can show the spectrum of the sizes and abundance of TERRA RNA. However, the strong secondary structure of TERRA could impede their migration properties in agarose gels, and there is a dilemma for the transfer of short vs long TERRA molecules. It has been proved that if alkaline treatment is omitted after electrophoresis, long TERRA molecules didn’t transfer [Beneden AV, 2013]. But treating the gels with alkaline cause small-sized RNA degradation and releasing from the gel, making quantifications rather difficult. Moreover, Northernblotting can only be used in the cells with abundant TERRA expression due to the low sensitivity of this detection method, which requires 5-10 ug RNA, but low abundant signal still couldn't be visualized. Dot-blotting can provide the information of overall TERRA repeat abundance, and quantification of TERRA expression, but information of TERRA length is missing. qRT-PCR measurement can detect low abundant TERRA and make the accurate quantification of the numbers of TERRA molecules, but the signals are from short sequences in the subtelomeric region of TERRA transcript, it couldn't be used for the analysis for TERRA length regulation.
For the detection of TERRA repeat length, Porro et al. reported the mUUAGGG tract-specific reverse transcription (Porro A, 2010). Cellular RNA was polyadenylated in vitro with recombinant yeast Papl, then was reverse transcribed with a mixture of six oligonucleotides that were predicted to hybridize to the junction between the UUAGGG sequence in all six permutated registers and the poly(A). RT was first carried out in the presence of only three nucleotides ([32P]dCTP, dTTP, and dATP) in order to restrict reverse transcription to the pure UUAGGG tract. The 1-h pulse was followed by a chase reaction in which an excess of unlabeled dCTP and, for the dGTP reaction, dGTP was added in order to allow RT beyond the cytosine-containing subtelomeric TERRA sequence. We applied this RT method to detect the length differences of TERRA repeat in supt4 siRNA treated cells. The positive control was successful, showing the majority of the shorter products were chased into longer cDNAs with dGTP, indicating dGTP is required to reverse transcribe beyond cytosine bases (data not shown). However, the differences of UUAGGG repeat length couldn’t be visualized in the presence of only three nucleotides ([32P]dCTP, dTTP, and dATP) (data not shown). The reason might be nucleotide variation of telomere repeat. This experimental approach assumes that human telomeric tracts are made of pure TTAGGG repeats, which may not be a general feature of telomeres. If there is a single nucleotide in the TERRA repeat tract is C, the reverse transcriptase can’t go through.
V. ADDITIONAL DISCUSSION
Different from most cancers which rely on the reactivation of telomerase for immortal replication, ALT cancers utilize BIR (break-induced replicate ALT mechanisms on) -based mechanism for telomere elongation and are usually associated with poorer prognosis. The ALT-mediated telomere elongation requires production of TERRA, an RNA transcribed using telomeric DNA as a template. In humans, the DNA template consists at least in part of repeats of a DNA sequence six nucleotides in length. TERRA is a crucial factor in triggering BIR and promoting telomere elongation in certain types of cancer. We have discovered that SUPT4H1 , a gene that encodes one of the DSIF (DRB sensitivity-inducing factor) elongation factor subunits, is required for the transcription of TERRA in ALT-dependent cancer cells and interference with Spt4h production can decrease TERRA transcript production and length, and decrease of Supt4h can lead to shortening of telomeres in cancer cells grown in culture. Associated with telomere shortening during cell culture is increased apoptotic cell death.
Cancer cells having replication associated with elevation of telomerase are not dependent on the ALT mechanisms. However, telomerase-dependent preservation of telomere length involved telomeric DNA containing nucleotide repeats.
SUPT4H1/5H, a highly conserved transcription elongation complex, can regulate RNA polymerase II processivity. SUPT4H1 has been reported as one of the key factors for the expression of long trinucleotide repeat containing transcripts in multiple disease models. Our investigations disclosed here indicate that SUPT4H1 is required for efficient transcription of TERRA, a hexanucleotide repeat (TTAGGG)6 non-coding RNA transcribed from the chromosome telomere region. The depletion of SUPT4H1 in U2OS, which is an osteosarcoma cell line harboring extremely long TERRA due to the mechanism of Alternative Lengthening of Telomere, has resulted in induced cell apoptosis due to the decreasing expression of TERRA and the consequent shortening of telomeres. Our findings suggest that SUPT4H1 may be a target gene in treating ALT cancers.
We have also discovered that telomerase activity in non-ALT cancer cells is reduced relative to total protein and also relative to nonspecific protein controls by interference with Supt4h. It thus appears that reduced Sujpt4h may interfere with the telomerase preservation of telomere length. As such, Supt4h functioning in transcription through nucleotide repeats of telomeres may be a target also for treatment of non-ALT cancers because of Supt4h effects on telomerase activity.
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Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e. , any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A method of treating a subject for a cellular proliferative disease, the method comprising: administering to the subject an agent that modulates DSIF complex activity in a manner sufficient to treat the subject for the cellular proliferative disease.
2. The method according to Claim 1 , wherein the DSIF complex comprises an SPT4 protein and an SPT5 protein.
3. The method according to Claim 2, wherein the DSIF complex comprises Supt4h and Supt5h.
4. The method according to any of the preceding claims, wherein the agent inhibits activity of a SPT4 protein, inhibits binding of an SPT4 protein to a second protein and/or inhibits expression of an SPT4 protein.
5. The method according to Claim 4, wherein the agent inhibits activity of a SPT4 protein.
6. The method according to Claim 4, wherein the agent inhibits binding of a SPT4 protein to a second protein.
7. The method according to Claim 4, wherein the agent inhibits expression of a SPT4 protein.
8. The method according to any of the preceding claims, wherein the cellular proliferative disease comprises a cancer.
9. The method according to Claim 8, wherein the cancer comprises an ALT+ cancer.
10. The method according to any of the preceding claims, wherein the subject is a human.
11. A method of reducing expression of a nucleotide repeat region that is not present in a gene in a cell, the method comprising: introducing into the cell an agent that modulates DSIF complex activity in a manner sufficient to reduce expression of the nucleotide repeat region that is not present in a gene.
12. The method according to Claim 11 , wherein the DSIF complex comprises an SPT4 protein and an SPT5 protein.
13. The method according to Claim 2, wherein the DSIF complex comprises Supt4h and Supt5h.
14. The method according to any of the preceding claims, wherein the agent inhibits activity of a SPT4 protein, inhibits binding of an SPT4 protein to a second protein and/or inhibits expression of an SPT4 protein.
15. The method according to Claim 14, wherein the agent inhibits activity of a SPT4 protein.
16. The method according to Claim 14, wherein the agent inhibits binding of a SPT4 protein to a second protein.
17. The method according to Claim 14, wherein the agent inhibits expression of a SPT4 protein.
18. The method according to any of Claims 11 to 17, wherein the nucleotide repeat region comprises a telomeric nucleotide repeat.
19. The method according to Claim 18, wherein the telomeric nucleotide repeat region comprises TTAGGG repeats.
20. The method according to any of Claims 11 to 19, wherein the cell is present in a multicellular subject.
21 . The method according to Claim 20, wherein the method is a method of treating the subject for a cellular proliferative disease.
22. The method according to Claim 21 , wherein the cellular proliferative disease comprises a cancer.
23. The method according to Claim 22, wherein the cancer comprises an ALT+ cancer.
24. The method according to any of Claims 20 to 23, wherein the subject is a human.
PCT/US2023/033228 2022-09-21 2023-09-20 Treatment of cellular proliferative disease via dsif complex modulation, and compositions for practicing the same WO2024064192A1 (en)

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