Patent Application
20220135971
GBMs are heterogeneous tumors that arise from astrocytes—the star-shaped cells that make up the “glue-like” or supportive tissue of the brain. Glioblastomas usually contain a mix of cell types. It is not unusual for these tumors to contain cystic mineral, calcium deposits, blood vessels, or a mixed grade of cells, and are nourished by an ample blood supply. Recent advances in treatment for patients with glioblastoma (GBM) have produced only a modest survival benefit with few long-term survivors. New effective and safe therapies are urgently needed to enhance outcomes for GBM patients.
Provided are compositions and methods for treating cancer. In one aspect, the cancer is a glioblastoma (GBM).
In one embodiment, a method of reprogramming an astrocyte to a glioblastoma stem-like cell (GSC) by introducing at least one master regulator selected from the group consisting of: NKX2-2, ETV4, MLXIPL, MEOX2, PRKCB, DDN, or OTP into a cell.
In another embodiment, a method of inhibiting a glioblastoma stem-like cell (GSC) by administering an immunotherapy composition that inhibits or reduces the expression of at least one master regulator selected from the group consisting of: NKX2-2, ETV4, MLXIPL, MEOX2, PRKCB, DDN, or OTP.
In another aspect, a method of treating a subject for glioblastoma by administering an immunotherapy composition that inhibits or reduces the expression of at least one master regulator selected from the group consisting of: NKX2-2, ETV4, MLXIPL, MEOX2, PRKCB, DDN, or OTP.
In another embodiment, an immunotherapy composition for treating a subject with a glioblastoma, comprising an inhibitor of at least one of NKX2-2, ETV4, MLXIPL, MEOX2, PRKCB, DDN, or OTP.
Having thus described the invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale.
A “master regulator” or “cancer master regulator” is a gene or protein that acts to drive one or more intermediary gene or proteins in a pathway or network important in initiating or maintaining a cancerous state or initiating or maintaining one or more deleterious cancerous behaviors. Some master regulators are involved in pathways in the transition to a cancer state.
A “master regulator network” refers to a master regulator and one or more genes downstream of the master regulator whose transcription level is dependent on or affected by the master regulator.
The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, refer to polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms include polymers that have been modified, such as polypeptides having modified peptide backbones.
Proteins are said to have an “N-terminus” and a “C-terminus.” The term “N-terminus” relates to the start of a protein or polypeptide, terminated by an amino acid with a free amine group (—NH2). The term “C-terminus” relates to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (—COOH).
The terms “nucleic acid” and “polynucleotide,” used interchangeably herein, refer to polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.
Nucleic acids are said to have “5′ ends” and “3′ ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5′ phosphate of one mononucleotide pentose ring is attached to the 3′ oxygen of its neighbor in one direction via a phosphodiester linkage. An end of an oligonucleotide is referred to as the “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the “3′ end” if its 3′ oxygen is not linked to a 5′ phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5′ and 3′ ends. In either a linear or circular DNA molecule, discrete elements are referred to as being “upstream” or 5′ of the “downstream” or 3′ elements.
“Codon optimization” refers to a process of modifying a nucleic acid sequence for enhanced expression in particular host cells by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. For example, a polynucleotide encoding a fusion polypeptide can be modified to substitute codons having a higher frequency of usage in a given host cell as compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, at the “Codon Usage Database.” The optimal codons utilized by L. monocytogenes for each amino acid are shown US 2007/0207170, herein incorporated by reference in its entirety for all purposes. These tables can be adapted in a number of ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, herein incorporated by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available (see, e.g., Gene Forge).
“Sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
“Percentage of sequence identity” refers to the value determined by comparing two optimally aligned sequences (greatest number of perfectly matched residues) over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.
Unless otherwise stated, sequence identity/similarity values refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof “Equivalent program” includes any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
The term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue. Typical amino acid categorizations are summarized below.
A “homologous” sequence (e.g., nucleic acid sequence) refers to a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence.
The term “fragment” when referring to a protein means a protein that is shorter or has fewer amino acids than the full length protein. The term “fragment” when referring to a nucleic acid means a nucleic acid that is shorter or has fewer nucleotides than the full length nucleic acid. A fragment can be, for example, an N-terminal fragment (i.e., removal of a portion of the C-terminal end of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminal end of the protein), or an internal fragment. A fragment can also be, for example, a functional fragment or an immunogenic fragment.
The term “in vitro” refers to artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube).
The term “in vivo” refers to natural environments (e.g., a cell or organism or body) and to processes or reactions that occur within a natural environment.
Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients.
Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.
Unless otherwise apparent from the context, the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value or variations ±0.5%, 1%, 5%, or 10% from a specified value.
The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an antigen” or “at least one antigen” can include a plurality of antigens, including mixtures thereof.
Statistically significant means p≤0.05.
Various embodiments of the inventions now will be described more fully hereinafter, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. The term “or” is used herein in both the alternative and conjunctive sense, unless otherwise indicated. The terms “illustrative” and “exemplary” are used to be examples with no indication of quality level.
Glioblastoma (GBM) is the most common and lethal form of adult human brain cancers. GBMs are formed by GBM stem-like cells (GSCs)—a major contributor to tumor recurrence and a natural focus for therapeutic development. There are two main reasons responsible for treatment failure: 1) high cellular and molecular heterogeneity; 2) GSCs have multiple redundant pathways requiring simultaneous targeting.
Details regarding various embodiments are described herein. By way of background, GBM is enriched in GBM stem-like cells (GSCs), a major contributor to tumor recurrence. Both GSCs and normal neuronal precursor cells (NPC) have the ability to form neurospheres when cultured in stem cell conditions. However, only GSCs can regenerate all cancer cells in the tumor when implanted in vivo (e.g., in vivo tumorigenicity). GSCs also can differentiate into other cells of the brain, however these cells are often not functional compared to those produced by NPCs. In a mouse model of GBM, elimination of self-renewal by genetic means led to a loss of GSCs and prolonged survival. However, as with other cancers, targeting GSCs has been a challenge because of the dearth of master regulators specific only to GSCs and not to NPCs or normal brain cells. The cell origin of GSCs remains unclear; both NPCs and normal astrocytes (NA) have been shown to contribute to GSCs. As a result, several survival and growth signals in GSCs share parallels in NPCs and NAs, increasing potential toxicity for therapies that target these pathways. Many of these targets are downstream signaling nodes with overlapping functions, allowing them to compensate for one another's blockade. Another challenge is the high intra- and inter-tumor heterogeneity in the GSC compartment, which necessitates the development of therapies that can target most, if not all, fractions of different subclones within and across many tumors. Recent genomics studies suggest that like other cancers, GBM originates from a founding GSC clone that emerged after sustaining a series of initiating and cooperative alterations that are passed on such that all subclones contain the founding alterations (i.e., the core common master regulators) and hence are targetable. As the number of potential founding alterations is surprisingly small, many founding alterations are expected to be common across different tumors of the same type or even of different types.
Founding alterations may produce “imprints” on the global gene regulatory network that may persist as the founding clone morphs into subclones and may be traceable across subclones. However, understanding the biological implications of these genomic alterations requires novel analytic tools that interrogate large-scale gene expression profiles to provide information on cancer cell's behaviors caused by interactions between the founding alterations and the tumor microenvironment. Gene expression profiles can then be used to infer the global and local networks that control such behaviors. This can be achieved using reverse engineering tools such as ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks), designed to scale up to the complexity of mammalian cells. ARACNe applies a theoretical information approach to infer gene networks using gene expression data, by calculating Mutual Information (MI).
In some embodiments, two computational engines GeneRep and nSCORE are applied to optimize the use of ARACNe and to quantitatively rank master regulators in any network, respectively. This strategy is greatly enhanced by the coupling with a multi-pronged compound-screening scheme.
GeneRep and nSCORE address two difficulties in computational biology: how to set a threshold cutoff level to maximize sensitivity while minimizing the false discovery rate (FDR) and how to incorporate various ranking parameters known individually to influence network hierarchy. GeneRep employs innovative coupling of bootstrapping with a random networks generation procedure from the real data. Networks generated at the gene level by GeneRep contain 20,000 nodes, while those generated at the transcript level contain 50,000 nodes. The number of edges ranges from 300,000 to 1 million, far higher than what is often obtained with current methods. nSCORE creates an automated node importance scoring framework that incorporates limitless sets of existing parameters and thus can be applied to any type of networks and node statistics inputs. GeneRep-nSCORE is described in WO-2018/069891, which is incorporated by reference in its entirety.
The master regulator identification and targeting workflow integrates key aspects to optimize success: GeneRep-nSCORE to rapidly identity GSC-specific master regulators at apices of signaling networks; intra- and inter-tumor heterogeneity analyses to identify master regulators common among GSC subclones; mutational and survival analyses to capture additional relevant master regulators; a two-pronged compound screening platform combining in silico and ultra-high throughput functional screens; evaluation of the clinical timeframe from surgery to drug identification; and development of a quantitative, network-based predictive biomarker for treatment response in GSCs.
We previously elucidated the roles of BASP1, NKX6.2, STOX2, MYCN, SOX8, OLIG2, HES6, and ASCL1 in reprogramming AST to GSC in WO 2018/211409, which is incorporated herein in its entirety.
Here we disclose further genes that play a role in reprogramming AST to GSC.
Additionally, we take a closer look at a subset of master regulators involved in reprograming astrocytes to GSCs, i.e., MEOX2, PRKCB, DDN, ETV4, MLXIPL, and OTP in combination with ASCL1, BASP1, MYCN, NKX6-2, and SOX8 (
NKX2-2 (NK2 Homeobox 2) encodes a protein that contains a homeobox domain and may be involved in the morphogenesis of the central nervous system. Diseases associated with NKX2-2 include Maturity-Onset Diabetes Of The Young and Cranial Nerve Malignant Neoplasm. Among its related pathways are Developmental Biology and Embryonic and Induced Pluripotent Stem Cell Differentiation Pathways and Lineage-specific Markers.
MEOX2 (Mesenchyme Homeobox 2) is a protein coding gene. Diseases associated with MEOX2 include Female Stress Incontinence and Low Compliance Bladder. Gene Ontology (GO) annotations related to this gene include DNA-binding transcription factor activity and RNA polymerase II proximal promoter sequence-specific DNA binding.
PRKCB (Protein Kinase C Beta) is a member of the protein kinase C (PKC) family of serine- and threonine-specific protein kinases that can be activated by calcium and second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This protein kinase has been reported to be involved in many different cellular functions, such as B cell activation, apoptosis induction, endothelial cell proliferation, and intestinal sugar absorption. Studies in mice also suggest that this kinase may also regulate neuronal functions and correlate fear-induced conflict behavior after stress.
DDN (Dendrin) is a protein coding gene. The DDN protein has been associated with promoting apoptosis of kidney glomerular podocytes.
ETV4 (ETS Variant Transcription Factor 4) is a protein coding gene. Diseases associated with ETV4 include Ewing Sarcoma and Extraosseous Ewing Sarcoma. Among its related pathways are RET signaling and Transcriptional misregulation in cancer.
MLXIPL (MLX Interacting Protein Like) encodes a basic helix-loop-helix leucine zipper transcription factor of the Myc/Max/Mad superfamily. This protein forms a heterodimeric complex and binds and activates, in a glucose-dependent manner, carbohydrate response element (ChoRE) motifs in the promoters of triglyceride synthesis genes. The gene is deleted in Williams-Beuren syndrome, a multisystem developmental disorder caused by the deletion of contiguous genes at chromosome 7q11.23.
OTP (Orthopedia Homeobox) encodes a member of the homeodomain (HD) family. HD family proteins are helix-turn-helix transcription factors that play key roles in the specification of cell fates.
In embodiments, a method of reprogramming an astrocyte to a glioblastoma stem-like cell (GSC) by introducing at least one master regulator selected from the group consisting of: NKX2-2, ETV4, MLXIPL, MEOX2, PRKCB, DDN, or OTP into a cell. In embodiments, a method of reprogramming an astrocyte to a glioblastoma stem-like cell (GSC) by introducing at least one master regulator selected from the group consisting of: NKX2-2, ETV4, MLXIPL, MEOX2, PRKCB, DDN, or OTP and further comprising introducing at least one master regulator from the group consisting of: BASP1, NKX6.2, STOX2, MYCN, SOX8, OLIG2, HES6, and ASCL1 into a cell.
In embodiments, a method of reprogramming an astrocyte to a glioblastoma stem-like cell (GSC) by introducing at least two master regulators selected from the group consisting of: NKX2-2, ETV4, MLXIPL, MEOX2, PRKCB, DDN, or OTP and further comprising introducing at least one master regulator from the group consisting of: BASP1, NKX6.2, STOX2, MYCN, SOX8, OLIG2, HES6, and ASCL1 into a cell. In embodiments, a method of reprogramming an astrocyte to a glioblastoma stem-like cell (GSC) by introducing at least 3, 4, 5, 6, or 7 master regulators selected from the group consisting of: NKX2-2, ETV4, MLXIPL, MEOX2, PRKCB, DDN, or OTP and further comprising introducing at least one master regulator from the group consisting of: BASP1, NKX6.2, STOX2, MYCN, SOX8, OLIG2, HES6, and ASCL1 into a cell.
In embodiments, a method of reprogramming an astrocyte to a glioblastoma stem-like cell (GSC) by introducing at least one master regulator selected from the group consisting of: NKX2-2, ETV4, MLXIPL, MEOX2, PRKCB, DDN, or OTP and further comprising introducing at least two master regulators from the group consisting of: BASP1, NKX6.2, STOX2, MYCN, SOX8, OLIG2, HES6, and ASCL1 into a cell. In embodiments, a method of reprogramming an astrocyte to a glioblastoma stem-like cell (GSC) by introducing at least one master regulator selected from the group consisting of: NKX2-2, ETV4, MLXIPL, MEOX2, PRKCB, DDN, or OTP and further comprising introducing at least 3, 4, 5, 6, 7, or 8 master regulators from the group consisting of: BASP1, NKX6.2, STOX2, MYCN, SOX8, OLIG2, HES6, and ASCL1 into a cell.
In embodiments, a method of reprogramming an astrocyte to a glioblastoma stem-like cell (GSC) by introducing NKX2-2, ETV4, MLXIPL, MEOX2, PRKCB, DDN, or OTP and further comprising introducing BASP1, NKX6.2, STOX2, MYCN, SOX8, OLIG2, HES6, and ASCL1 into a cell.
The presently disclosed subject matter provides master regulators, such as NKX2-2, MEOX2, PRKCB, DDN, ETV4, MLXIPL, and OTP that when inhibited, can reduce or inhibit GSCs. In some embodiments, inhibition of at least one of these master regulators can be used to inhibit GSCs. In some embodiments, inhibition of a combination of at least two of these master regulators can be used to inhibit GSCs. In some embodiments, inhibition of at least one of these master regulators can be used to treat a subject with glioblastoma. In some embodiments, a combination of inhibition of at least two of these master regulators can be used to treat a subject with glioblastoma. In some embodiments, the presently disclosed subject matter provides a method of reprogramming normal human astrocytes to GSCs by introducing a combination of the master regulators disclosed herein into a cell. In some embodiments, inhibition of a combination of the master regulators NKX2-2, MEOX2, PRKCB, DDN, ETV4, MLXIPL, and OTP can be used to inhibit GSCs or in therapeutic methods for treating glioblastoma.
In some embodiments, a method of inhibiting GSCs or treating glioblastoma comprising using or administering an immunotherapy composition against individual or combinations of the master regulators disclosed herein. Also provided are immunotherapy compositions that target at least one of the master regulators disclosed herein In one embodiment, the immunotherapy composition comprises a peptide formulation derived from at least one of the master regulators disclosed herein. In one embodiment, the immunotherapy composition comprises nanoparticle or dendritic cell containing peptides derived from at least one of the master regulators disclosed herein. In one embodiment, the immunotherapy composition comprises RNAs coding for at least one of the master regulators disclosed herein. In one embodiment, the immunotherapy composition comprises nanoparticles or dendritic cells containing RNAs coding for at least one master regulator disclosed herein. In one embodiment, the RNAs coding for master regulators are electroporated into dendritic cells.
Also provided are pharmaceutical compositions that inhibit at least one master regulator disclosed herein. In one embodiment, the inhibitor is a RNA interference agent or a small molecule.
In one embodiment, delivery of the composition is by direct injection into the brain. In one embodiment, delivery is by gene therapy, for example by adeno-associated virus (AAV) or retroviral replication vector (RRV) vector. In one embodiment, delivery is by systemic intravenous delivery.
In some embodiments, we describe methods of treating cancer comprising inhibiting one or more master regulators. Inhibiting one or more master regulators can comprise using or administering one or more master regulator antagonists or inhibitors. A master regulator can be inhibited at the gene level, such as by using or administering RNA interference agents or antisense oligonucleotides to inhibit expression of the gene. The master regulators can be inhibited at the protein level, such as by using or administering an immunotherapy composition that binds to the master regulator protein and inhibits activity of the protein or by using or administering a small molecule drug known to inhibit activity of the master regulator protein. In some embodiments, we described methods of treating cancer comprising using or administering an immunotherapy composition against a master regulator protein or a combination of master regulator proteins. An immunotherapy composition can comprise one or more antibodies having affinity for one or more master regulators. An antibody can be, but is not limited to, an immunoglobulin, an immunoglobulin fragment having affinity for the master regulator, a chimeric antibody, a bispecific antibody, an antibody conjugate, or the like.
In some embodiments, an immunotherapy composition comprises a peptide formulation derived from a master regulator. The peptide can be an immunogenic fragment of a master regulator protein. The peptide can be combined with an immune stimulating adjuvant. The immunotherapy composition can be administered locally (e.g., subcutaneously) or systemically (e.g., intravenously) with or without the presence of adjuvant. The immunotherapy composition can be used to stimulate the immune system to develop an immune reaction specifically against the master regulator. Development of an immune reaction can eliminate or aid in eliminating cancer cells expressing the master regulator.
In some embodiments, we describe methods of treating cancer comprising using or administering one or more small molecule drugs to inhibit activity of a master regulator protein or a combination of master regulator proteins. In embodiments, the method comprises administering immunotherapy compositions, small molecules, RNA interference agents, antisense oligonucleotides, or combinations thereof that target one or more of the master regulators associated with the cancer.
In some embodiments, we describe methods of treating cancer comprising using or administering one or more antisense oligonucleotides or RNA interference agents to knock down expression of a master regulator gene or a combination of master regulator genes. An antisense oligonucleotide is a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid. An RNA interference agent is an oligonucleotide that mediates the targeted cleavage of an RNA transcript in a sequence specific manner via an RNA-induced silencing complex (RISC) pathway.
In some embodiments, we describe methods of treating cancer comprising using or administering a combination of one or more master regulator antagonists or inhibitors.
In one embodiment, the master regulator is NKX2-2. In one embodiment, NKX2-2 has the sequence of SEQ ID No: 2 or NG 042186.1. In one embodiment, a method of treating a cancer or tumor by administering and inhibitor of NKX2-2 to a subject in need thereof. In one embodiment, the inhibitor that targets NKX2-2 targets SEQ ID No: 2 or NG 042186.1 or a fragment thereof.
In one embodiment, the master regulator is MLXIPL. In one embodiment, MLXIPL has the sequence of SEQ ID Nos: 4, 6, 8, 10, or NG 009307.1. In one embodiment, a method of treating a cancer or tumor by administering and inhibitor of MLXIPL to a subject in need thereof. In one embodiment, the inhibitor that targets MLXIPL targets SEQ ID Nos: 4, 6, 8, 10, NG 009307.1, or a fragment thereof.
In one embodiment, the master regulator is ETV4. In one embodiment, ETV4 has the sequence of SEQ ID No: 12, 14, 16, 18, 20, or NC_000017.11. In one embodiment, a method of treating a cancer or tumor by administering and inhibitor of ETV4 to a subject in need thereof. In one embodiment, the inhibitor that targets ETV4 targets SEQ ID No: 12, 14, 16, 18, 20, NC_000017.11, or a fragment thereof.
In one embodiment, the master regulator is MEOX2. In one embodiment, MEOX2 has the sequence of SEQ ID No: 22 or NG_032988.1. In one embodiment, a method of treating a cancer or tumor by administering and inhibitor of MEOX2 to a subject in need thereof. In one embodiment, the inhibitor that targets MEOX2 targets SEQ ID No: 22 or NG_032988.1 or a fragment thereof.
In one embodiment, the master regulator is PRKCB. In one embodiment, PRKCB has the sequence of SEQ ID No: 24 or 26 or NG_029003.2. In one embodiment, a method of treating a cancer or tumor by administering and inhibitor of PRKCB to a subject in need thereof. In one embodiment, the inhibitor that targets PRKCB targets SEQ ID No: 24 or 26 or NG_029003.2 or a fragment thereof.
In one embodiment, the master regulator is DDN. In one embodiment, DDN has the sequence of SEQ ID No: 28 or NC_000012.12. In one embodiment, a method of treating a cancer or tumor by administering and inhibitor of DDN to a subject in need thereof. In one embodiment, the inhibitor that targets DDN targets SEQ ID No: 28 or NC_000012.12 or a fragment thereof.
In one embodiment, the master regulator is OTP. In one embodiment, OTP has the sequence of SEQ ID No: 30 or NC_000005.10. In one embodiment, a method of treating a cancer or tumor by administering and inhibitor of OTP to a subject in need thereof. In one embodiment, the inhibitor that targets OTP targets SEQ ID No: 30 or NC_000005.10 or fragment thereof.
In one embodiment, a method of treating a subject with a cancer or tumor comprising administering a composition comprising an inhibitor of at least one master regulator disclosed herein. In one embodiment, the master regulator is selected from the group consisting of NKX2-2, MEOX2, PRKCB, DDN, ETV4, MLXIPL, and OTP.
In one embodiment, a method of treating a subject with a cancer or tumor. In one embodiment, the cancer or tumor is a glioblastoma. In one embodiment, the tumor is a glioma. In one embodiment, the tumor is from brain. In one embodiment, the cancer or tumor is non-small cell lung cancer or cancer where the cell type of origin are from neurodectoderm.
There are two different populations of GSCs, slow-cycling and fast-cycling. Slow-cycling GSCs are slow-dividing but they give rise to fast-cycling GSCs, which are fast-dividing. Fast-cycling GSC are more susceptible to therapeutics since they are target fast-dividing. Therefore, targeting the slow-cycling GSCs will destroy the tumor since slow-cycling GSCs replenish the fast-dividing GSCs which are dying off due to cancer therapeutics.
Here, we explore the GSCs master regulators NKX6.2 and ASCL1 and whether expression is specific to regulating slow-cycling GSCs or fast-cycling GSCs or both. We show that NKX6.2 preferentially expressed and is essential for slow cycling GSCs, but not fast cycling GSCs (
Master regulators are genes at the top of a gene network which can alter the expression of downstream genes in a network. Applying the tandem computational platform GeneRep-nSCORE that integrates large-scale gene expression profiles with genomic changes to identify common founding master regulators of GSCs spanning across most, if not all, GSC clones, we discovered set of common master regulators in GCSs that are outstanding targets for clinical development.
We applied the GeneRep-nSCORE platform to gene expression profiles of GSCs and GBM differentiating cells (GDC), normal neuronal precursor cells (NPC), and normal human astrocytes (NHA) and predicted top genes involved in fate conversions between these cell types.
Here, we take a closer look at a subset of the master regulators: MEOX2, PRKCB, ETV4, along with NKX6-2.
We used lentiviruses encoding for shRNA specific for one or two master regulators and transduced 3 independent patient-derived GSC lines. These results confirmed that effective inhibition of one or two master regulators, either by genetic means (si/shRNA) or perhaps small molecule inhibitors, would have significant therapeutic potential as a GSC-specific treatment of GBM, and possibly for other cancers whose stem cells share similar regulatory pathways.
These experiments were performed in 3 individual patient derived GSC cell lines (CA7, R24-03, or R24-01) and to the same result. Together, these findings show that these master regulators may serve as important pharmacologically targets that and may reduce tumorigenicity (i.e., reduced tumor size or number of tumors).
Combinations of MEOX2 and PRKCB, MEOX2 and ETV4, MEOX2 and NKX6.2, and ASCL1 and NKX6.2 were tested in vivo in mice.
We depleted different combinations of master regulators using lentiviral shRNA in xenograft tumors in mice. The control shRNA contained a scrambled sequence. These xenografts were derived from several GSC lines that have been labeled with a bioluminescent.
Recurrent tumors in experimental mice grew from cells that did not have master regulators depleted. This shows that efficient depletion is crucial.
Results are shown in
The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5′ end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3′ end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which the inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
This invention was made with government support K08CA160824 awarded by National Institute of Health (NIH). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
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| PCT/US20/17090 | 2/6/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62802554 | Feb 2019 | US |
