Target genes in MYC-driven neoplasia

Methods are provided for treating a subject having a MYC-driven neoplasia. Aspects of the methods include administering to the subject an amount of an inhibitor of a target gene effective to treat the subject for the MYC-driven neoplasia. Methods are also provided for identifying a MYC-dependent target gene in a MYC-driven neoplasia. Aspects of the method include identifying the MYC-dependent target gene based on a phenotype detected in a first tumor cell line conditionally expressing MYC that is absent or quantitatively different in a second tumor cell line conditionally repressing MYC when the two cell lines are contacted with a CRISPR-based gene silencing agent. Kits and cell lines for practicing the methods of the disclosure are also provided.

A Sequence Listing is provided herewith as a text file, “STAN-1359WO_SeqList_ST25.txt” created on Dec. 20, 2017 and having a size of 1,161 KB. The contents of the text file are incorporated by reference herein in their entirety.

BACKGROUND

MYC is a master transcription factor that can regulate the expression of up to 15% of genes in the genome1,2. Overexpression of MYC is thought to contribute to the pathogenesis of over 50% of human cancers3. Experimentally, the inhibition of MYC reverses tumorigenesis in transgenic mouse models4-6. Therapeutically targeting MYC would have broad clinical impact across multiple cancer types. However, identifying small molecules that directly target MYC has been extremely challenging7,8. The structure of MYC lacks any druggable domains, and as a result it has been impossible to design compounds that cleanly hit the protein9.

An alternative approach to directly targeting MYC is to target genes that are specifically relied upon by MYC-addicted cancers, but otherwise do not result in adverse effects within cells not addicted to MYC, such as non-cancer cells. Attempts to identify such targets, referred to as synthetic lethals, have previously employed methods of screening that rely on incomplete inhibition of target genes, such as RNA interference (RNAi).

The identification of additional druggable targets, for example, through the use of improved methods of screening for synthetic lethal targets in MYC-driven cancers that result in complete inhibition of target genes, will increase the development of effective cancer therapeutics. Furthermore, treating subjects having cancers that are specifically identified as MYC-addicted with such new therapeutics circumvents the issues associated with the undruggability of MYC. This approach specifically takes advantage of the very element of these cancers that significantly contributes to pathogenesis, namely MYC overexpression.

Publications

SUMMARY

Methods are provided for treating a subject having a MYC-driven neoplasia. Aspects of the methods include administering to the subject an amount of an inhibitor of a target gene effective to treat the subject for the MYC-driven neoplasia. Methods are also provided for identifying a MYC-dependent target gene in a MYC-driven neoplasia. Aspects of the method include identifying the MYC-dependent target gene based on a phenotype detected in a first tumor cell line conditionally expressing MYC that is absent or quantitatively different in a second tumor cell line conditionally repressing MYC when the two cell lines are contacted with a CRISPR-based gene silencing agent. Kits and cell lines for practicing the methods of the disclosure are also provided.

DEFINITIONS

The terms “specific binding,” “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides). In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD(dissociation constant) of 10−5M or less (e.g., 10−6M or less, 10−7M or less, 10−8M or less, 10−3M or less, 10−10M or less, 10−11M or less, 10−12M or less, 10−13M or less, 10−14M or less, 10−15M or less, or 10−16M or less). “Affinity” refers to the strength of binding, increased binding affinity being correlated with a lower KD.

The terms “antibody” and “immunoglobulin”, as used herein, are used interchangeably may generally refer to whole or intact molecules or fragments thereof and modified and/or conjugated antibodies or fragments thereof that have been modified and/or conjugated. The immunoglobulins can be divided into five different classes, based on differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a given class will have very similar heavy chain constant regions. These differences can be detected by sequence studies or more commonly by serological means (i.e. by the use of antibodies directed to these differences). Immunoglobulin classes include IgG (Gamma heavy chains), IgM (Mu heavy chains), IgA (Alpha heavy chains), IgD (Delta heavy chains), and IgE (Epsilon heavy chains).

Antibody or immunoglobulin may refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized, see for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region typically is comprised of three domains, CH1, CH2, and CH3. Each light chain typically is comprised of a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region typically is comprised of one domain, CL. The VHand VLregions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).

Whole or largely intact antibodies are generally multivalent, meaning they may simultaneously bind more than one molecule of antigen whereas antibody fragments may be monovalent. Antibodies produced by an organism as part of an immune response are generally monospecific, meaning they generally bind a single species of antigen. Multivalent monospecific antibodies, i.e. antibodies that bind more than one molecule of a single species of antigen, may bind a single antigen epitope (e.g., a monoclonal antibody) or multiple different antigen epitopes (e.g., a polyclonal antibody).

Multispecific (e.g., bispecific) antibodies, which bind multiple species of antigen, may be readily engineered by those of ordinary skill in the art and, thus, may be encompassed within the use of the term “antibody” used herein where appropriate. Also, multivalent antibody fragments may be engineered, e.g., by the linking of two monovalent antibody fragments. As such, bivalent and/or multivalent antibody fragments may be encompassed within the use of the term “antibody”, where appropriate, as the ordinary skilled artisan will be readily aware of antibody fragments, e.g., those described below, which may be linked in any convenient and appropriate combination to generate multivalent monospecific or polyspecific (e.g., bispecific) antibody fragments.

Antibody fragments include but are not limited to antigen-binding fragments (Fab or F(ab), including Fab′ or F(ab′), (Fab)2, F(ab′)2, etc.), single chain variable fragments (scFv or Fv), “third generation” (3G) molecules, etc. which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind to the subject antigen, examples of which include, but are not limited to:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule;

(3) (Fab)2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction;

(4) F(ab)2is a dimer of two Fab′ fragments held together by two disulfide bonds;

(5) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains;

(6) Single chain antibody (“SCA”), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule; such single chain antibodies may be in the form of multimers such as diabodies, triabodies, tetrabodies, etc. which may or may not be polyspecific (see, for example, WO 94/07921 and WO 98/44001) and

(7) “3G”, including single domain (typically a variable heavy domain devoid of a light chain) and “miniaturized” antibody molecules (typically a full-sized Ab or mAb in which non-essential domains have been removed).

The terms “treatment”, “treating”, “treat” and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. The term “treatment” encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom(s) but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting development of a disease and/or the associated symptoms; or (c) relieving the disease and the associated symptom(s), i.e., causing regression of the disease and/or symptom(s). Those in need of treatment can include those already inflicted (e.g., those with cancer, e.g. those having tumors) as well as those in which prevention is desired (e.g., those with increased susceptibility to cancer; those with cancer; those suspected of having cancer; etc.).

The terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is human.

A “therapeutically effective amount”, a “therapeutically effective dose” or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy, achieve a desired therapeutic response, etc.). A therapeutically effective dose can be administered in one or more administrations. For purposes of this disclosure, a therapeutically effective dose of an agent that inhibits a target gene (e.g., a MYC-dependent target gene, and the like) and/or compositions is an amount that is sufficient, when administered to the individual, to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease state (e.g., cancer, etc.) by, for example, inhibiting the growth of, inducing death of or otherwise preventing the clinical progressing of a MYC-dependent cancer present in the subject.

DETAILED DESCRIPTION

Methods are provided for treating a subject having a MYC-driven neoplasia. Aspects of the methods include administering to the subject an amount of an inhibitor of a target gene effective to treat the subject for the MYC-driven neoplasia. Methods are also provided for identifying a MYC-dependent target gene in a MYC-driven neoplasia. Aspects of the method include identifying the MYC-dependent target gene based on a phenotype detected in a first tumor cell line conditionally expressing MYC that is absent or quantitatively different in a second tumor cell line conditionally repressing MYC when the two cell lines are contacted with a CRISPR-based gene silencing agent. Kits and cell lines for practicing the methods of the disclosure are also provided.

Methods

As summarized above, methods of the present disclosure include methods of identifying a MYC-dependent target gene. Aspects of such methods generally include contacting two neoplastic cell lines (e.g., two cancer cell lines, two tumor cell lines, etc.), one conditionally expressing MYC and one conditionally repressing MYC, with a CRISPR-based gene silencing agent targeting a target gene and identifying one or more phenotypes differentially induced in the two different neoplastic cell lines. Accordingly, the subject methods identify target genes that, when targeted with a CRISPR-based gene silencing agent, result in a MYC-dependent phenotype. A MYC-dependent phenotype may be correlated with the gene targeted by the CRISPR-based gene silencing agent, allowing the identification of the targeted gene as a MYC-dependent target gene.

MYC-dependent phenotypes will vary and, in some instances, may include MYC-dependent death of a cell or cells of a MYC-expressing neoplastic cell line. MYC-dependent death of a cell or cells of the MYC-expressing neoplastic cell line may be determined in any convenient method including e.g., by comparison with the rate of cell death or viability/survival of a cell or cells of a corresponding MYC-repressing neoplastic cell line. In instances where the identified MYC-dependent phenotype includes increased cell death or decreased viability of the cells expressing MYC, as compared to control cells where MYC is repressed, the identified phenotype may be referred to as a synthetic lethal phenotype. Such synthetic lethal phenotypes will generally include where the reduced expression of a target gene, through targeting with a CRISPR-based gene silencing agent, results in decreased viability of MYC expressing cells but does not (or insignificantly) results in a decrease in viability of cells which do not express MYC, repress MYC or otherwise express MYC at a level below that of the MYC expressing cells. Target genes identified as resulting in a MYC-dependent synthetic lethality may be referred to as MYC-dependent synthetic lethal target genes.

By “MYC-dependent target gene”, as used herein, is meant a gene, targeted by a CRISPR-based gene silencing agent, which results in a MYC-dependent phenotype. In some instances, identified MYC-dependent target genes may be further targeted in the treatment of a subject, as described in more detail below, utilizing various agents that target the target gene, including e.g., agents that target the activity of the target gene, agents that specifically bind an encoded product of the target gene to inhibit its activity, agents that inhibit the expression of the target gene, and the like. Accordingly, the term “target gene” as used herein may refer to the targeting of the CRISPR-based gene silencing agent to the target gene and/or the targeting of a therapeutic to the target gene or an expression product thereof.

Methods of the present disclosure may be employed to identify synthetic lethal interactions with MYC thereby identifying MYC-synthetic lethal genes. An identified MYC-synthetic lethal gene may be a MYC-dependent target gene. The expression of MYC-dependent target genes, as defined herein, may or may not be directly or indirectly controlled by MYC. For example, in some instances, a MYC-dependent target gene may be a direct target of MYC such that MYC directly controls the expression of the target gene. In some instances, a MYC-dependent target gene may be an indirect target of MYC such that MYC indirectly controls the expression of the target gene. In some instances, a MYC-dependent target gene may not be a direct or indirect target of MYC such that MYC does not directly or indirectly control the expression of the target gene. Such a target gene having expression that is not directly or indirectly controlled by MYC may nonetheless represent a MYC synthetic lethal interaction and be a MYC-synthetic lethal gene that is identified by the methods described herein. Accordingly, the MYC-synthetic lethal genes and the MYC-dependent target genes identified using the methods described herein will not be limited to genes conventionally identified as targets of MYC (i.e., MYC target genes) and will include both genes that are and are not direct and/or indirect targets of MYC (i.e., MYC target genes).

By “CRISPR-based gene silencing agent” is meant one or more agents that when delivered to a cell cause the directed silencing of a target gene by CRISPER/Cas9-based nuclease activity. Accordingly, in some instances, a CRISPR-based gene silencing agent may include a guide RNA (gRNA) having sequence that specifically targets a Cas9 nuclease to a specific target gene. CRISPR/Cas9-based silencing of a target gene may include delivery of a Cas9 polypeptide or a Cas9 polypeptide encoding nucleic acid to the subject cells. For example, in some instances, a vector that includes a nucleic acid that encodes a Cas9 nuclease may be delivered to the subject cells before, during or after the cell is contacted with a CRISPR-based gene silencing agent such that the encoded Cas9 nuclease is expressed when the CRISPR-based gene silencing agent is present within the cell. In some instances, the cell may be genetically modified with a nucleic acid encoding a Cas9 nuclease such that the encoded Cas9 nuclease is expressed (e.g., conditionally expressed, constitutively expressed, etc.) when the CRISPR-based gene silencing agent is present within the cell. Accordingly, CRISPR/Cas9-based silencing of the present methods may employ a Cas9 nuclease that is stably or transiently expressed including e.g., where a nucleic acid encoding the Cas9 nuclease is transiently or stably present within the cell line. In some instances, Cas9 polypeptide may be delivered to the subject cells, i.e., without the need to express the Cas9 polypeptide within the cells. CRISPR-based gene silencing agents will vary and may include e.g., vector (e.g., virus (e.g., lentivirus), plasmid, etc.) containing and/or expressing one or more gRNAs. Methods of delivery of CRISPR-based gene silencing agents will similarly vary any may include e.g., transfection, electroporation, lipofection, etc.

CRISPR-based gene silencing agents of the present disclosure may be directed to essentially any element of a subject genome including e.g., protein-coding and non-protein coding elements of the subject genome. In some instances, e.g., where a plurality of CRISPR-based gene silencing agents is employed, the plurality of CRISPR-based gene silencing agents may collectively target all or essentially all genes of the subject genome (i.e., genome-wide targeting). In some instances, targeted non-protein coding elements may include but are not limited to e.g., promoters, enhancers, non-coding RNAs, and the like. In some instances, the targets of one or more CRISPR-based gene silencing agents may include proteins involved in RNA metabolism and/or nucleic acids encoding proteins involved in RNA metabolism. Proteins involved in RNA metabolism include but are not limited to e.g., proteins involved in RNA transcription amplification, mRNA splicing, ribosomal biogenesis, RNA transport, RNA degradation and the like. In some instances, the targets of one or more CRISPR-based gene silencing agents may include proteins involved in processes other than RNA metabolism including but not limited to e.g., DNA repair, pyrimidine metabolism, terpenoid backbone biosynthesis, and the like.

In some instances, a phenotype identified may be a susceptibility to a cancer therapy, including e.g., a MYC-dependent susceptibility to a cancer therapy. A MYC-driven neoplasm having a MYC-dependent susceptibility to a cancer therapy may be more susceptible to the cancer therapy than the corresponding neoplasm that lacks or displays reduced MYC expression (including e.g., where the MYC expression is conditionally controlled). By “cancer therapy”, in the instant context, is meant any convenient cancer therapy including but not limited to e.g., radiation therapy, chemotherapy, immunotherapy, and the like.

As noted above, the subject methods of identifying a MYC-dependent target gene will generally include contacting a first cell line and a second cell line with the CRISPR-based gene silencing agent where the first and second cell lines differ in their expression of MYC. The first and second cell lines will generally be sufficiently similar to allow for a meaningful comparison, including e.g., where the first and second cell lines are identical except for their expression of MYC. In some instances, the first and second cell lines may be identical (i.e., derived from the same parental cell line) and may differ in their expression of MYC only by the presence or absence of an agent that induces or represses expression of MYC. For example, in some instances, a cell line useful in the subject methods may be a cell line that conditionally expresses or repress MYC in the presence of tetracycline or an analog thereof. In some instances, the first and second cell lines may only differ in that one has been contacted with an agent that induces or represses MYC expression in the cell line (e.g., tetracycline or an analog thereof) and the other has not been contacted with the agent. In such instances, tetracycline or any analog thereof may be employed.

The subject methods of identifying a MYC-dependent target gene may be performed in multiplex fashion, including e.g., where contacting the cell lines with a CRISPR-based gene silencing agent targeting a target gene may include contacting the cell lines with a plurality of CRISPR-based gene silencing agents targeting a plurality of different target genes. Such pluralities of target genes will vary and may include e.g., where the plurality includes all or essentially all of the genes of a genome of a subject such as a mammal (e.g., a mouse, rat, primate, human, etc.). In some instances, a plurality of target genes may include only the genes of a particular functional group including e.g., genes involved in RNA metabolism. In some instances, a plurality of CRISPR-based gene silencing agents may be referred to as a library of CRISPR-based gene silencing agents and contacting cell lines, as described herein, may include contacting the cell line with the library.

As summarized above, the methods of the present disclosure include treating a subject for a MYC-driven neoplasia by administering to the subject an effective amount (e.g., a therapeutically effective amount) of an agent that inhibits a MYC-dependent target gene or the expression product thereof. Such methods may include administering to a subject one or more agents that inhibit a MYC-dependent target gene identified utilizing the methods of CRISPR/Cas9-based screening described herein. In some instances, the subject methods include administering to a subject an inhibitor of a MYC-dependent target gene identified according to the methods described herein as a MYC-dependent synthetic lethal target gene. Targets employed in the subject methods need not necessarily be limited to those identified in a subject screen and may generally include other targets that when inhibited preferentially treat MYC-driven neoplasia as compared to MYC-independent neoplasia.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of a MYC-dependent target gene and/or one or more inhibitors of an expression product of a MYC-dependent target gene selected from: AHR, AURKA, BIRC5, BRD4, CDK9, EP300, HMGCS1, MTOR, PIM3 and PRMT5.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of a protein involved in RNA metabolism, including but not limited to e.g., RNA transcription amplification, mRNA splicing, ribosomal biogenesis, RNA transport or RNA degradation.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of ribosome formation or activity including e.g., an inhibitor of RPS12, RPL37A, MRPL21, MRPL20, MRPL23, RPL7, RPLP1, RPL5, RPL35A, RPS15A, RPS6, RPS10, MRPL13, RPL36AL, MRPS21, RPS8, RPL26, RPL13A, RPS19, MRPL36, MRPL12, RPS25, RPS21, MRPS18A, RPS28, RPL3, RPL14, MRPL4, RPSA, RPL39, RPL32 or RPL31.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of a basal transcription factor including e.g., an inhibitor of TAF7, TAF13, TAF3, TAF2, TAF6L, GTF2F2, GTF2H3, TBP, ERCC2, ERCC3, GTF2B, TAF1, TAF11, TAF10 or CCNH.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of aminoacyl-tRNA biosynthesis including e.g., an inhibitor of QRSL1, KARS, IARS2, EARS2, NARS2, AARS, WARS2, WARS, MARS2, FARSB, RARS, DARS2, AARS2, LARS2, HARS or GATC.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of pyrimidine metabolism including e.g., an inhibitor of POLR1E, POLD1, ZNRD1, NME3, CMPK1, PNPT1, POLD2, TYMS, POLR1B, POLR1C, DTYMK, DHODH, RRM1, POLR2G, POLR2L, POLD3, POLR2J, DUT, POLR3K or CAD.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of the cell cycle including e.g., an inhibitor of HDAC2, ATR, CCNA2, ANAPC10, GADD45G, SMC1A, RAD21, STAG2, ORC4, ORC5, ORC3, MCM6, MCM5, MCM3, EP300, E2F3, E2F2, CCND1, CDC26, CDC45, CCNH or CREBBP.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of nucleotide excision repair including e.g., an inhibitor of GTF2H3, RFC4, ERCC2, ERCC3, RFC5, RFC2, POLD1, POLD2, POLD3, RPA3, DDB2, RPA2 or CCNH.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of spliceosome activity or formation including e.g., an inhibitor of NCBP1, SF3B5, SNRPA1, PRPF8, SRSF7, RBM8A, SRSF3, U2AF2, HNRNPU, SF3A1, SF3A2, SF3A3, TRA2B, LSM7, SMNDC1, LSM5, HNRNPA1, ALYREF, THOC1, SNRPF, SNRPD2 or ACIN1.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of DNA replication including e.g., an inhibitor of MCM6, RFC5, RFC4, MCM5, RPA3, RFC2, POLD1, POLD2, POLD3, MCM3 or RPA2.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of homologous recombination including e.g., an inhibitor of MRE11A, RPA3, RPA2, POLD1, POLD2, POLD3, RAD51, XRCC3 or TOP3A.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of mismatch repair, including e.g., an inhibitor of RFC5, RFC4, RPA3, RFC2, POLD1, POLD2, POLD3 or RPA2.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of the mRNA surveillance pathway, including e.g., an inhibitor of PPP2R1A, SMG7, SYMPK, NCBP1, DAZAP1, PPP2R2D, ACIN1, ALYREF, WIBG, UPF1, WDR82, PABPC1, RNGTT, CPSF3 or RBM8A.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of RNA degradation, including e.g., an inhibitor of DCPS, LSM7, PABPC1, PNPT1, CNOT10, C1D, ENO1, DIS3, XRN2, LSM5, EXOSC2 or EXOSC7.

In some embodiments, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of terpenoid backbone biosynthesis, including e.g., an inhibitor of MVD, HMGCS1, RCE1, IDI1 or NUS1.

In some instances, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering to the subject an effective amount of one or more inhibitors of a protein involved in RNA transport including but not limited to e.g., Acin1, Alyref, Eif3a/b/f, Eif5b, Ncbp1, Ndc1, Nups, Pabpc1, Rbm8a, Seh1l, Sumo2, Thoc1, Upf1, Wibg, Xpo1, Gemin2/7/8, Ncbp1, Ndc1, Nups, Prmt5, Seh1l, Smn1, Xpo1, Ndc1, Nups, Pop1/4/5/7, Rpp25l, Seh1l, Xpo1, Eef1a1, Ndc1, Nups, Rpp21, Seh1l and Trnt1. In some instances, the inhibitor of RNA transport inhibits one or more of mRNA transport, snRNA transport, rRNA transport and/or tRNA transport.

In some instances, the methods of the present disclosure include treating a subject for a MYC-driven cancer by administering the subject an effective amount of one or more inhibitors of a protein involved in DNA repair, pyrimidine metabolism, or terpenoid backbone biosynthesis.

Any useful inhibitor of the subject target gene and/or encoded product thereof may be employed in the subject methods. Non-limiting examples of useful inhibitors include but are not limited to e.g., non-peptide small molecule antagonists, peptide antagonists, interfering RNAs (e.g., siRNA, shRNA, etc.), antibodies (e.g., neutralizing antibodies, function blocking antibodies, etc.), aptamers, and the like. In some instances, inhibitors may target, e.g., specifically bind to, specifically hybridize to, etc., a target protein or a nucleic acid encoding a target protein including where the protein shares 100% sequence identity or less than 100% sequence identity, including e.g., at least 99%, at least 98%, at least 97% at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, etc., sequence identity, with a protein or amino acid sequence of a protein described herein. Accordingly, as non-limiting examples, in some instances, useful inhibitors may include a non-peptide small molecule antagonist of a protein involved in RNA metabolism, a peptide antagonist of a protein involved in RNA metabolism, an interfering RNA targeting an RNA expressed from a RNA metabolism gene, an anti-RNA-metabolism-protein antibody (e.g., an antibody that specifically binds to an RNA metabolism protein), an anti-RNA-metabolism-protein aptamer, and the like. In some instances, the effectiveness of an inhibitor may be confirmed using an in vitro or in vivo assay, including e.g., where the effectiveness of the inhibitor is compared to an appropriate control or standard, e.g., the corresponding CRISPR-based gene silencing agent, a conventional cancer therapy, etc.

An individual to be treated according to the present methods will generally be an individual with a neoplasia. As used herein “neoplasia” includes any form of abnormal new tissue formation; and the like. In some cases, the individual has recently undergone treatment for neoplasia (e.g., cancer, a tumor, etc.) and are therefore at risk for recurrence. In some instances, the individual has not recently or previously undergone treatment for a neoplasia (e.g., cancer, a tumor, etc.) but has been newly diagnosed with a neoplasia. Any and all neoplasia are suitable neoplasia to be treated by the subject methods e.g., utilizing a subject inhibitor of a MYC-dependent target gene or a herein described treatment kit. The subject methods will generally include the treatment of MYC-driven neoplasia, including e.g., malignant MYC-driven neoplasia including a MYC-driven cancer, a MYC driven tumor, and the like.

In some instances, a MYC-driven cancer treated according to the methods described herein is a blood cancer (e.g., a leukemia, a lymphoma, etc.), In some instances, a MYC-driven cancer treated according to the methods described herein is a liver cancer (e.g., a hepatocellular cancer, e.g., a hepatocellular carcinoma). In some instances, the neoplasia treated according to the methods described herein is not colon cancer or a tumor thereof. In some instances, the neoplasia treated according to the methods described herein is not breast cancer or a tumor thereof.

In some instances, the subject methods of treating a subject for a MYC-driven neoplasia may include a step of identifying the subject's neoplasia as MYC-driven. Any convenient method for identifying a neoplasia as MYC-driven may be employed including but not limited to e.g., measuring MYC expression in a sample of the neoplasia from the subject, detecting a mutation in the subject associated with MYC overexpression, and the like. Measuring MYC expression in a sample of the neoplasia from the subject may be performed in a variety of ways including but not limited to e.g., quantitative PCR, quantitative sequencing, microarray expression profiling, in situ hybridization, quantitative mass spectrometry, immunohistochemistry, and the like. Various mutations associated with MYC overexpression may be detected and such mutations will vary and will include any mutation useful in determining the resulting expression level of a MYC encoding nucleic acid, the expression level of a MYC polypeptide, and/or the activity or function of an encoded MYC polypeptide. Accordingly, mutations associated with MYC overexpression that may be detected will vary any may include but are not limited to e.g., gain-of-function mutations in MYC, mutations that inhibits the activity a MYC repressor, mutations that induce the activity of a MYC activator and the like.

The compositions (e.g., those including one or more inhibitor of a MYC-dependent target gene) of this disclosure can be supplied in the form of a pharmaceutical composition. Any suitable pharmaceutical composition may be employed, described in more detail below. As such, in some instances, methods of the present disclosure may include administering an inhibitor in a composition comprising an excipient (e.g., an isotonic excipient) prepared under sufficiently sterile conditions for administration to a mammal, e.g., a human.

Administration of an inhibitor to a subject, as described herein, may be performed employing various routes of administration. The route of administration may be selected according to a variety of factors including, but not necessarily limited to, the condition to be treated, the formulation and/or device used, the patient to be treated, and the like. Routes of administration useful in the disclosed methods include but are not limited to oral and parenteral routes, such as intravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic, nasal, and transdermal. Formulations for these dosage forms are described herein.

An effective amount of a subject compound will depend, at least, on the particular method of use, the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. A “therapeutically effective amount” of a composition is a quantity of a specified compound sufficient to achieve a desired effect in a subject (host) being treated.

Therapeutically effective doses of a subject compound or pharmaceutical composition can be determined by one of skill in the art, with a goal of achieving local (e.g., tissue) concentrations that are at least as high as the IC50 of an applicable compound disclosed herein.

The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the subject compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex and diet of the subject, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the host undergoing therapy.

Conversion of an animal dose to human equivalent doses (HED) may, in some instances, be performed using the conversion table and/or algorithm provided by the U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) in, e.g.,Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers(2005) Food and Drug Administration, 5600 Fishers Lane, Rockville, Md. 20857; (available at www(dot)fda(dot)gov/cder/guidance/index(dot)htm, the disclosure of which is incorporated herein by reference).

Conversion of Animal Doses to Human Equivalent Doses Based on Body Surface Area

To Convert AnimalTo Convert Animal Dose in mg/kgDose in mg/kg toto HEDain mg/kg, Either:Dose in mg/m2,Divide AnimalMultiplySpeciesMultiply by kmDose ByAnimal Dose ByHuman37——Child (20 kg)b25——Mouse312.30.08Hamster57.40.13Rat66.20.16Ferret75.30.19Guinea pig84.60.22Rabbit123.10.32Dog201.80.54Primates:Monkeysc123.10.32Marmoset66.20.16Squirrel75.30.19monkeyBaboon201.80.54Micro-pig271.40.73Mini-pig351.10.95aAssumes 60 kg human. For species not listed or for weights outside the standard ranges, HED can be calculated from the following formula: HED = animal dose in mg/kg × (animal weight in kg/human weight in kg)0.33.bThis km value is provided for reference only since healthy children will rarely be volunteers for phase 1 trials.cFor example, cynomolgus, rhesus, and stumptail.

Pharmaceutical Compositions

A pharmaceutical composition comprising a subject compound (i.e., an inhibitory agent or a combination thereof) may be administered to a patient alone, or in combination with other supplementary active agents. The pharmaceutical compositions may be manufactured using any of a variety of processes, including, without limitation, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, and lyophilizing. The pharmaceutical composition can take any of a variety of forms including, without limitation, a sterile solution, suspension, emulsion, lyophilisate, tablet, pill, pellet, capsule, powder, syrup, elixir or any other dosage form suitable for administration.

A subject compound may be administered to the host using any convenient means capable of resulting in the desired reduction in disease condition or symptom. Thus, a subject compound can be incorporated into a variety of formulations for therapeutic administration. More particularly, a subject compound can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

Formulations for pharmaceutical compositions are well known in the art. For example, Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995, describes exemplary formulations (and components thereof) suitable for pharmaceutical delivery of disclosed compounds. Pharmaceutical compositions comprising at least one of the subject compounds can be formulated for use in human or veterinary medicine. Particular formulations of a disclosed pharmaceutical composition may depend, for example, on the mode of administration and/or on the location of the infection to be treated. In some embodiments, formulations include a pharmaceutically acceptable carrier in addition to at least one active ingredient, such as a subject compound. In other embodiments, other medicinal or pharmaceutical agents, for example, with similar, related or complementary effects on the affliction being treated can also be included as active ingredients in a pharmaceutical composition.

Pharmaceutically acceptable carriers useful for the disclosed methods and compositions are conventional in the art. The nature of a pharmaceutical carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can optionally contain minor amounts of non-toxic auxiliary substances (e.g., excipients), such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like; for example, sodium acetate or sorbitan monolaurate. Other non-limiting excipients include, nonionic solubilizers, such as cremophor, or proteins, such as human serum albumin or plasma preparations.

The disclosed pharmaceutical compositions may be formulated as a pharmaceutically acceptable salt of a disclosed compound. Pharmaceutically acceptable salts are non-toxic salts of a free base form of a compound that possesses the desired pharmacological activity of the free base. These salts may be derived from inorganic or organic acids. Non-limiting examples of suitable inorganic acids are hydrochloric acid, nitric acid, hydrobromic acid, sulfuric acid, hydroiodic acid, and phosphoric acid. Non-limiting examples of suitable organic acids are acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, methyl sulfonic acid, salicylic acid, formic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, asparagic acid, aspartic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and the like. Lists of other suitable pharmaceutically acceptable salts are found in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pa., 1985. A pharmaceutically acceptable salt may also serve to adjust the osmotic pressure of the composition.

A subject compound 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. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles. Formulations suitable for injection can be administered by an intravitreal, intraocular, intramuscular, subcutaneous, sublingual, or other route of administration, e.g., injection into the gum tissue or other oral tissue. Such formulations are also suitable for topical administration.

In some embodiments, a subject compound can be delivered by a continuous delivery system. The term “continuous delivery system” is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.

A subject compound can be utilized in aerosol formulation to be administered via inhalation. A subject compound can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, a subject compound can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. A subject compound can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

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 a subject compound calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for a subject compound depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

The dosage form of a disclosed pharmaceutical composition will be determined by the mode of administration chosen. For example, in addition to injectable fluids, topical or oral dosage forms may be employed. Topical preparations may include eye drops, ointments, sprays and the like. In some instances, a topical preparation of a medicament useful in the methods described herein may include, e.g., an ointment preparation that includes one or more excipients including, e.g., mineral oil, paraffin, propylene carbonate, white petrolatum, white wax and the like, in addition to one or more additional active agents.

Oral formulations may be liquid (e.g., syrups, solutions or suspensions), or solid (e.g., powders, pills, tablets, or capsules). Methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

Certain embodiments of the pharmaceutical compositions comprising a subject compound may be formulated in unit dosage form suitable for individual administration of precise dosages. The amount of active ingredient administered will depend on the subject being treated, the severity of the affliction, and the manner of administration, and is known to those skilled in the art. Within these bounds, the formulation to be administered will contain a quantity of the extracts or compounds disclosed herein in an amount effective to achieve the desired effect in the subject being treated.

Each therapeutic compound can independently be in any dosage form, such as those described herein, and can also be administered in various ways, as described herein. For example, the compounds may be formulated together, in a single dosage unit (that is, combined together in one form such as capsule, tablet, powder, or liquid, etc.) as a combination product. Alternatively, when not formulated together in a single dosage unit, an individual subject compound may be administered at the same time as another therapeutic compound or sequentially, in any order thereof.

Kits and Cell Lines

Also provided are kits and cell lines for use in the subject methods. The subject cell lines include neoplastic cell lines (e.g., tumor cell lines, cancer cell lines, etc.) conditionally expressing and/or repressing MYC. In some instances, the subject cell lines conditionally express and/or repress MYC in the presence of tetracycline or an analog of tetracycline, such as but not limited to e.g., doxycycline. Cell lines may include those derived from a MYC-driven neoplasia including e.g., those MYC-driven neoplasms derived from a mammalian neoplasia such as e.g., a human neoplasia, a mouse neoplasia, a rat neoplasia, a primate neoplasia, or the like. In some instances, the MYC-driven neoplasia may be derived from a model organism neoplasia, including MYC-driven model organism neoplasia, e.g., those derived from a mouse model organism neoplasia, a rat model organism neoplasia, a primate model organism neoplasia, or the like. In some embodiments, the subject cell line may be a liver cancer cell line. In some embodiments, the subject cell line may be a blood cancer cell line. In some instances, the cell line may be a carcinoma cell line including but not limited to a liver carcinoma cell line, such as a MYC-drive liver carcinoma cell line. In some instances, the cell line may be a lymphoma cell line or a leukemia cell line such as but not limited to e.g., a MYC-driven Burkitt's lymphoma/leukemia cell line, a MYC-driven T cell lymphoma/leukemia cell line, a MYC-driven B cell lymphoma/leukemia cell line, or the like.

The subject kits may include any combination of components (e.g., reagents, cell lines, etc.) for performing the subject methods, such as e.g., methods of treating a subject for a neoplasm and/or methods of identifying a target gene of a MYC-driven neoplasm.

In some embodiments, a subject kit may be employed in a method of treating a subject for a MYC-driven neoplasm. Such a kits may vary and may, but need not necessarily, include one or more reagents for identifying the neoplasm as a MYC-driven neoplasm and an effective amount of an agent for treating the MYC-driven neoplasm including but not limited to e.g., one or more inhibitors of RNA metabolism (e.g., an inhibitor of RNA transcription amplification, an inhibitor of mRNA splicing, an inhibitor of ribosomal biogenesis, an inhibitor RNA degradation, an inhibitor of RNA transport, or a combination thereof), one or more inhibitors of DNA repair, one or more inhibitors of pyrimidine metabolism, one or more inhibitors of terpenoid backbone biosynthesis, or a combination thereof. In some instances, a kit may include one or more inhibitors of the following RNA transport genes: Acin1, Alyref, Eif3a/b/f, Eif5b, Ncbp1, Ndc1, Nups, Pabpc1, Rbm8a, Seh1l, Sumo2, Thoc1, Upf1, Wibg, Xpo1, Gemin2/7/8, Ncbp1, Ndc1, Nups, Prmt5, Seh1l, Smn1, Xpo1, Ndc1, Nups, Pop1/4/5/7, Rpp25l, Seh1l, Xpo1, Eef1a1, Ndc1, Nups, Rpp21, Seh1l and Trnt1. Therapeutic agents provided in such kits may or may not be configured as a pharmaceutical formulation, in unit dosage form, alone or in combination, e.g., as described in more detail herein.

In some embodiments, a subject kit may be employed in a method of identifying a target gene of a MYC-driven neoplasm. Such kits may vary and may, but need not necessarily, include one or more tumor cell lines that conditionally express/repress MYC in response to the presence/absence of tetracycline or an analog thereof. In some instances, a subject kit may include one or more, including a plurality of or a library of, CRISPR-based gene silencing agents. In some embodiments, the subject kits may include a nucleic acid for expressing a Cas9 polypeptide within a tumor cell line that conditionally expresses/represses MYC in the presence of tetracycline or an analog thereof. In some instances, a cell line contained within a subject kit may be configured (e.g., genetically modified) to express a Cas9 polypeptide.

In addition to the above components, the subject kits and/or cell lines may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits or provided with the subject cell lines in a variety of forms, one or more of which may be present in the kit or provided with the subject cell lines. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit or cell line(s), in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.

Notwithstanding the appended claims, the disclosure is also defined by the following clauses:

1. A method of identifying a MYC-dependent target gene, the method comprising:

a) contacting a first tumor cell line, conditionally expressing MYC, with a CRISPR-based gene silencing agent targeting a target gene;

b) contacting a second tumor cell line, conditionally repressing MYC, with the CRISPR-based gene silencing agent; and

c) detecting a phenotype of the first tumor cell line that is present in or quantitatively different in the second tumor cell line to identify the target gene as a MYC-dependent target gene.

2. The method according to Clause 1, wherein the first and second tumor cell lines conditionally repress MYC in the presence of tetracycline or an analog thereof.

3. The method according to Clauses 1 or 2, wherein the phenotype is present in the first tumor cell line and absent in the second tumor cell line.

4. The method according to Clause 3, wherein the phenotype is cell death.

5. The method according to Clause 3, wherein the phenotype is susceptibility to a cancer therapy.

6. The method according to Clauses 1 or 2, wherein the phenotype is quantitatively decreased in the first tumor cell line as compared to the second tumor cell line.

7. The method according to Clause 6, wherein the phenotype is cell viability.

8. The method according to any of the preceding clauses, wherein the first and second tumor cell lines are carcinoma cancer cell lines.

9. The method according to any of the preceding clauses, wherein the first and second tumor cell lines are liver cancer cell lines.

10. The method according to any of the preceding clauses, wherein the first and second tumor cell lines transiently express a Cas9 nuclease.

11. The method according to any of the preceding clauses, wherein the first and second tumor cell lines stably express a Cas9 nuclease.

12. The method according to any of the preceding clauses, wherein the CRISPR-based gene silencing agent is a guide RNA (gRNA).

13. The method according to any of the preceding clauses, wherein the target gene encodes a protein involved in RNA metabolism.

14. The method according to Clause 13, wherein the protein involved in RNA metabolism is a protein involved in RNA transcription amplification, mRNA splicing, ribosomal biogenesis, RNA transport or RNA degradation.

15. The method according to any of the preceding clauses, wherein the target gene is an RNA transport gene selected from the group consisting of: Acin1, Alyref, Eif3a/b/f, Eif5b, Ncbp1, Ndc1, Nups, Pabpc1, Rbm8a, Seh1l, Sumo2, Thoc1, Upf1, Wibg, Xpo1, Gemin2/7/8, Ncbp1, Ndc1, Nups, Prmt5, Seh1l, Smn1, Xpo1, Ndc1, Nups, Pop1/4/5/7, Rpp25l, Seh1l, Xpo1, Eef1a1, Ndc1, Nups, Rpp21, Seh1l and Trnt1.
16. The method according to any of Clauses 1 to 12, wherein the target gene encodes a protein involved in DNA repair, pyrimidine metabolism, or terpenoid backbone biosynthesis.
17. The method according to any of the preceding clauses, wherein the method comprises contacting the first and second tumor cell lines with a plurality of CRISPR-based gene silencing agents targeting a plurality of target genes and detecting one or more phenotypes of the first tumor cell line that are present in or quantitatively different in the second tumor cell line to identify the plurality of target genes as MYC-dependent target genes.
18. A method of treating a subject for a neoplasia that is MYC-driven, the method comprising administering to the subject an effective amount of an inhibitor of a MYC-dependent target gene identified according to the method of any of Clauses 1 to 17.
19. The method according to Clause 18, wherein the method further comprises identifying the neoplasia as a MYC-driven neoplasia.
20. The method according to Clause 19, wherein the identifying comprises measuring MYC expression in a sample of the neoplasia from the subject.
21. The method according to Clauses 19 or 20, wherein the identifying comprises detecting a mutation in the subject associated with MYC overexpression.
22. The method according to Clause 21, wherein the mutation is a gain-of-function mutation in MYC.
23. The method according to Clause 21, wherein the mutation is a mutation that inhibits the activity a MYC repressor.
24. The method according to Clause 21, wherein the mutation is a mutation that induces the activity of a MYC activator.
25. The method according to any of Clauses 18 to 24, wherein the neoplasia is a neoplasia of the liver or blood.
26. The method according to any of Clauses 18 to 25, wherein the neoplasia is a carcinoma.
27. The method according to any of Clauses 18 to 25, wherein the neoplasia is a lymphoma.
28. A method of treating a subject for neoplasia that is MYC-driven, the method comprising administering to the subject an effective amount of an inhibitor of RNA metabolism.
29. The method according to Clause 28, wherein the inhibitor of RNA metabolism is an inhibitor of RNA transcription amplification.
30. The method according to Clause 28, wherein the inhibitor of RNA metabolism is an inhibitor of mRNA splicing.
31. The method according to Clause 28, wherein the inhibitor of RNA metabolism is an inhibitor of ribosomal biogenesis.
32. The method according to Clause 28, wherein the inhibitor of RNA metabolism is an inhibitor RNA degradation.
33. The method according to Clause 28, wherein the inhibitor of RNA metabolism is an inhibitor of RNA transport.
34. The method according to Clause 33, wherein the inhibitor of RNA transport is an inhibitor of Xpo1.
35. The method according to Clause 34, wherein the inhibitor of Xpo1 is KPT-330.
36. The method according to any of Clauses 28 to 35, wherein the method further comprises identifying the neoplasia as a MYC-driven neoplasia.
37. The method according to Clause 36, wherein the identifying comprises measuring MYC expression in a sample of the neoplasia from the subject.
38. The method according to Clauses 36 or 37, wherein the identifying comprises detecting a mutation in the subject associated with MYC overexpression.
39. The method according to any of Clauses 28 to 38, wherein the neoplasia is a neoplasia of the liver or blood.
40. The method according to any of Clauses 28 to 39, wherein the neoplasia is a carcinoma.
41. The method according to any of Clauses 28 to 39, wherein the neoplasia is a lymphoma.
42. A method of treating a subject for a neoplasia that is MYC-driven, the method comprising administering to the subject an effective amount of an inhibitor of AHR, AURKA, BIRC5, BRD4, CDK9, EP300, HMGCS1, MTOR, PIM3 or PRMT5.
43. The method according to Clause 42, wherein the neoplasia is a neoplasia of the liver or blood.
44. The method according to Clause 42 or 43, wherein the neoplasia is a carcinoma.
45. The method according to Clause 42 or 43, wherein the neoplasia is a lymphoma.
46. The method according to any of Clauses 42 to 45, wherein the method further comprises identifying the neoplasia as a MYC-driven neoplasia.
47. The method according to Clause 46, wherein the identifying comprises measuring MYC expression in a sample of the neoplasia from the subject.
48. The method according to Clauses 46 or 47, wherein the identifying comprises detecting a mutation in the subject associated with MYC overexpression.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., room temperature (RT); base pairs (bp); kilobases (kb); picoliters (pl); seconds (s or sec); minutes (m or min); hours (h or hr); days (d); weeks (wk or wks); nanoliters (nl); microliters (ul); milliliters (ml); liters (L); nanograms (ng); micrograms (ug); milligrams (mg); grams ((g), in the context of mass); kilograms (kg); equivalents of the force of gravity ((g), in the context of centrifugation); nanomolar (nM); micromolar (uM), millimolar (mM); molar (M); amino acids (aa); kilobases (kb); base pairs (bp); nucleotides (nt); intramuscular (i.m.); intraperitoneal (i.p.); subcutaneous (s.c.); and the like.

Example 1: CRISPR Synthetic Lethal Screen Identifies RNA Transport as a Novel Therapeutic Target for MYC Overexpressing Cancer

Materials and Methods

CRISPR Library Screening and Data Analysis

The CRISPR library screening was performed following published protocol14. The pooled mouse GeCKO v2 CRISPR library containing 130,209 gRNAs was obtained from Addgene. Briefly, the library was amplified by electroporation into the Endura competent cells (Lucigen). After lentiviral packaging of the library, EC4 cells with Cas9 were infected at low viral titers so that approximately 10% of the cells were infected. The infected cells were selected with puromycin (1 μg/ml) for two days before screening.

The gRNA libraries in the screened populations were subsequently isolated by PCR amplification and characterized by hi-seq. Computational extraction of the gRNA from the fastq files was done by removing the barcode, stagger, and primer sequences, and aligning to the GeCKO libraries. Bowtie2 alignment used default parameters. Files were converted to bam format and sorted using samtools. These results were filtered to only include forward-aligning reads without indels to minimize the possibility of miscounting successful alignments. Reads passing filter were counted by reference contig (i.e. the 20mers in the GeCKO libraries) to give counts for each gRNA. Data were plotted using R/gplots.

KPT-330 Treatment In Vivo and Monitoring the Tumor Size

The KPT-330 (Selleckchem) was dissolved in vehicle (1% Pluronic F-68 and 1% PVP-K29/32, 25 mg/kg) was administered by oral gavage to tumor-bearing mice three times a week for 6 doses total. A 7T MRI for small animals was used to image tumor both before and after treatment. Tumor volume was calculated based on the T2 weighted image stacks using the Osirix software. For short-term treatment, the mice were treated with either vehicle or KPT-330 for three doses and sacrificed the following day after the third dose.

mRNA Fluorescent In Situ Hybridization

A standard FISH protocol was used to detect poly(A) in fixed cells. In brief, cells were fixed in 4% formaldehyde in PBS at room temperature for 20 minutes, washed 5 minutes each in PBS and 2×SSC (20×SSC: 3 M sodium chloride, 300 mM sodium citrate, pH=7.0) before applying the probe. The mRNA-specific probe (5′-/5ATTO565N/d(T)30-3′, from Integrated DNA Technologies) was hybridized at 37 degree for 3h in the dark (200 nM probe in 4×SCC, 0.5 mM EDTA, 10% dextran sulfate, and 10% formamide). Cells were then washed in 2×SSC and PBS (15 minutes each) before mounting. PBS, SSC, and water were treated with 0.1% diethylpyrocarbonate (DEPC) prior use. Images were acquired on a DMI 6000 B (Leica) epifluorescence microscope.

EU-Labeling and Visualization of Total RNA

EC4 cells were labeled with 1 mM 5-ethynyl uridine (EU) for 5h in the presence of 150 nM KPT-330 or vehicle (DMSO control) at 37 degrees and 5% CO2. Cells were then fixed with 4% formaldehyde in PBS at room temperature for 20 minutes, washed consecutively with PBS, 50 mM NH4Cl/PBS, PBS (10 minutes each) and permeabilized with 0.2% TritonX100/PBS. After washing with PBS, EU was labeled with Alexa-647-azide using the Click-IT imaging kit (Invitrogen) following the manufacturer's instructions. Images were acquired on a DMI 6000 B (Leica) epifluorescence microscope. Mean fluorescence intensities in regions of interest in the cytoplasm and in the nucleus omitting nucleoli were determined using ImageJ.

RNA Fractionation and RNA-Seq

EC4 cells were treated with either DMSO or KPT-330 (0.5 μM) in triplicates for 24 hours. RNA was fractionated using the Cytoplasmic & Nuclear RNA Purification kit (Norgen Biotek). RNA-seq was performed commercially by Macrogen. Fragments Per Kilobase of transcript per Million (FPKM) mapped reads were used to measure the abundance of each transcript. For the existing RNA-seq studies, the data were downloaded from Gene Expression Omnibus (GEO). The specific data sets are GSE76062 for primary murine LAP-tTA/tet-O-MYC liver cancer, GSE51008 for Eμ-MYC lymphoma, and GSE40783 for the human P493-6 cell line. The abundance of 34 RNA transport genes and B2M was normalized with the level of ubiquitin C (UBC).

Flow Cytometric Analysis of Apoptosis

For apoptosis staining, cells were stained with 7-AAD and PE-Annexin-V (Becton Dickinson) and analyzed on a FACScan flow cytometer following manufacturer's instructions. FACS data was analyzed with FlowJo software (Tree Star). The apoptotic cell populations were defined by positive staining of both Annexin-V and 7-AAD.

Western Blot and Immunohistochemical Analysis

The following antibodies were used for Western blotting and IHC: MYC (9E10 for Western blot and Epitomics 1472-1 for IHC), Tubulin (Sigma), Cleaved caspase 3 and phospho-Histone H3 (Cell Signaling). Immunofluorescence or bright field pictures were taken with 20× objectives on a Leica DMI6000 B microscope and quantified using MetaMorph image analysis software.

Cells were seeded at a density of 1000-2000 cells/well in 96-well plates. Cells were treated with different concentrations of KPT-330 (1:3 dilution from 10 micromolar to 0.33 nanomolar) for 72 hours. MYC expression was shut off by treating the cells with doxycycline (100ng/ml). All experiments were performed in quadruplicates. Cell viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). The IC50 was calculated using the Prism software (GraphPad). The log-transformed concentration values and the normalized luminance data were fitted to a four-parameter logistic equation to derive the IC50 value.

Results

CRISPR Genome-Wide Library Screen in a Conditional MYC Expressing Tumor

A CRISPR genome-wide library screen was performed in a conditional MYC expressing tumor cell line. First, a conditional MYC-induced cancer cell line (EC4) was derived from a Tet-regulated mouse model of hepatocellular carcinoma (LAP-tTA/tet-O-MYC)16. In the EC4 cell line, the human MYC transgene is overexpressed but its expression can be shut off by treatment with doxycycline. The Cas9 nuclease was introduced into EC4 cells and functionally confirmed to effectively induce mutations at specific genomic loci (FIG.5). To screen for synthetic lethal interactions with MYC (MYC-SL), 130,209 guide RNAs (gRNAs) targeting 20,611 mouse genes were delivered into the EC4 cells using lentiviral infection at low titers so that each cell contained at most one gRNA (FIG.1, Panel a)14. After selection, all surviving EC4 cells should contain one gRNA but mutations of genes targeted by the gRNAs have not yet occurred. These cells served as the baseline pool and were further passaged and separated into two pools: the SL pool that continued to overexpress MYC and the control pool that was treated with doxycycline to shut off MYC expression. Cells were maintained for one more week to allow for the accumulation of genomic mutations induced by the Cas9/gRNAs and the dropouts of specific gRNAs (FIG.1, Panel a). The frequency distribution of gRNAs in the baseline, SL, and control pools were examined with deep-seq. The overall gRNA frequency distribution in all three pools were similar (FIG.1, Panel b), suggesting that no significant drifting in the library representation had occurred during cell culture and screening. Furthermore, significant dropouts in the 1000 non-targeting gRNAs that serve as the internal control of the library screen were not observed.

The MYC-SL gene list was further analyzed for overrepresented molecular pathways which revealed that many pathways, such as ribosomal biogenesis, RNA transport, RNA transcription, spliceosome, aminoacyl-tRNA biosynthesis, pyrimidine metabolism, cell cycle and DNA replication, DNA repair, mRNA surveillance and RNA degradation, and terpenoid backbone biosynthesis are required by MYC overexpressing tumors (FIG.1, Panel d and Table 1). This analysis identified cellular processes that are essential in MYC-driven cancers.

Expression of the MYC-SL RNA Transport Genes is Upregulated by MYC

RNA metabolism pathways are the most notable and frequent changes identified in this screen as shown by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. For example, the screen identified multiple hits within cellular processes of RNA metabolism, such as ribosomal biogenesis13, aminoacyl-tRNA biosynthesis24, RNA transcription25-27, mRNA splicing12,22, RNA transport, and mRNA surveillance and degradation28(FIG.1, Panel d). An interesting key processes identified in this analysis, namely RNA transport, ranked as the second most significant changes by KEGG pathway analysis. RNA transport has not been exploited previously in MYC-driven cancers. 34 genes, representing 20% of all 171 genes involved in RNA transport, were identified in the screening hits (FIG.2, Panel a and Table 1). These 34 genes are involved in the processing and transport of multiple RNA species, such as mRNAs, snRNAs, rRNAs, and tRNAs (FIG.2, Panel a). This result suggests that the transport of various RNA species is a critical cellular process required by cancers overexpressing MYC.

Next, whether the expression of these 34 genes involved in RNA transport is regulated by MYC was examined. First, in the RNA-seq data sets of primary liver tumors derived from the LAP-tTA/tet-O-MYC mouse model16,29, MYC overexpression increased the transcript levels of these genes by 3-10 fold in liver tumors compared to normal liver tissues. Furthermore, upon the shutdown of MYC expression in liver tumors, the expression of these 34 genes significantly decreased within 16 hours to a level similar to that in normal liver tissues, suggesting that their expression is directly regulated by MYC activity (FIG.2, Panel b). As a control, the expression of β-2 microglobulin (B2m) did not change significantly. Second, in the Eμ-MYC lymphoma model30,31, a 2-7 fold steady increase in the expression of these genes was observed during the tumorigenesis from normal B cells to premalignant cancer cells, and then to overt lymphoma (FIG.2, Panel c). Lastly, in the human Burkitt lymphoma-like P493-6 cell line with Tet-regulated MYC expression32, MYC overexpression upregulates the expression of the RNA transport genes by 3-12 fold (FIG.2, Panel d). Thus, our analysis across multiple cancer contexts showed that these MYC-SL genes involved in RNA transport are regulated by MYC during tumorigenesis.

Xpo1 Inhibition Blocks RNA Export in MYC Overexpressing Cancer

Amongst the MYC-SL RNA transport genes, Prmt5 and Xpo1 are currently druggable and Xpo1 inhibitors are in clinical trials for multiple myeloma33-36. Xpo1 is a transport receptor that forms a complex with the Ran GTPase and drives the transport of multiple RNA species as well as protein targets37,38.

Whether Xpo1 inhibition can block RNA transportation in MYC-driven cancer cell lines was tested. KPT-330 is a highly selective small molecule chemical inhibitor of Xpo1 and a heterozygous point mutation of cysteine 528 in Xpo1 is sufficient to confer drug resistance to KPT-33039-41. Newly transcribed RNAs were labeled with a uridine analog 5-ethynyl uridine (EU) and detected the distribution of the labelled RNA in the cells with Click Chemistry42. The EC4 cancer cells treated with KPT-330 showed significantly reduced RNA distribution in the cytoplasm and increased RNA nuclear-cytoplasmic ratio, compared with the DMSO controls (FIG.3, Panels a-b). In contrast, the staining of α-tubulin and cellular DNA content did not differ between the control and the cells treated with KPT-330. These data suggest that Xpo1 inhibition significantly blocks the export of total RNA in tumor cells overexpressing MYC.

Xpo1 is a Therapeutic Target in MYC-Overexpressing Cancer

Next, whether MYC-overexpressing cancer cells are more sensitive to Xpo1 inhibition as compared to the low MYC expressing cells was evaluated. The EC4 cells were treated under either MYC ON or MYC OFF states. It was found that the EC4 cells with MYC overexpression are highly sensitive to Xpo1 inhibition with a four-fold induction of tumor cell apoptosis upon KPT-330 treatment, while the EC4 cells with MYC OFF only have a modest increase in apoptosis (FIG.4, Panel a). The half maximal inhibitory concentration (IC50) was further used to measure the sensitivity of KPT-330 in a panel of cell lines with high MYC expression (human Huh7, HepG2, and Hep40 liver cancer cell lines, human P493-6 Burkitt lymphoma-like cell line, a mouse MYC-driven T-cell acute lymphoblastic leukemia cell line, and an mouse IgH-MYC B-cell lymphoma cell line), liver cancer cell lines with low MYC expression (PLC/PRF/5, SNU-182, SNU-449)10,47, as well as the normal human fibroblast BJ5-tA cell line. The normal fibroblast cell line and the liver cancer cell lines with low MYC expression are not sensitive to Xpo1 inhibition with IC50s ranging from 0.3 μM to >10 μM. In contrast, MYC overexpressing cancer cell lines are sensitive to KPT-330 with the IC50s in the range of 0.07 μM to 0.3 μM (FIG.4, Panel b andFIG.6, Panel a). In both EC4 and P493-6 cells, downregulation of MYC expression also shifted up the IC50 curve, indicating that the tumor cells are more resistant to Xpo1 inhibition under low MYC expression (FIG.6, Panel b). Taken together, these data suggest that MYC overexpression is correlated with high sensitivity to Xpo1 inhibition.

It was further investigated whether Xpo1 inhibition can be an effective treatment for MYC-driven cancer in vivo. The therapeutic effect of Xpo1 inhibition was tested using primary liver tumors developed in the LAP-tTA/tet-O-MYC mouse model. MRI imagining was used to quantify the tumor volume both before and after KPT-330 treatment for two weeks. It was found that all the tumors drastically regressed upon Xpo1 inhibition with KPT-330 with a more than 95% reduction in tumor volume. In two mice with smaller tumors, complete tumor regression was observed according to the MRI scan (FIG.4, Panels c-e andFIG.7). In contrast, the tumor size increased 4-12 fold within the same two-week time frame in mice treated with vehicle control. To examine the mechanism of tumor regression, regressing tumors were isolated following a short-term Xpo1 inhibition, and tumor cell proliferation was studied by phospho-histone H3 staining and apoptosis by cleaved caspse-3 staining. Xpo1 inhibition completely suppressed the proliferation and significantly induced apoptosis in tumor cells (FIG.4, Panels f-g). Further histological examination of tumor sections showed only sporadic tumor cells left in mice with Xpo1 inhibition (FIG.4, Panel f andFIG.8). No significant proliferation or apoptosis was observed in the adjacent liver tissues in mice with Xpo1 inhibition, indicating minimal toxicity to the normal hepatocytes. These data demonstrate that tumors overexpressing MYC, compared to normal liver tissues, are more sensitive to Xpo1 inhibition and that Xpo1 is a highly effective druggable target in vivo.

DISCUSSION

Through a genome-wide CRISPR library screen, synthetic lethal interactions of MYC were identified with the goal of finding therapeutic targets for MYC-overexpressing cancers. It was found that multiple aspects of RNA metabolism, including RNA transcription amplification, mRNA splicing, ribosomal biogenesis, RNA transport, and RNA degradation, are essential for MYC-overexpressing cancers. In addition to RNA metabolism, it was also found that DNA repair, pyrimidine metabolism, and terpenoid backbone synthesis are required by MYC-overexpressing tumors.

Notably, this study showed that RNA transport, which is the intermediate step between RNA production in the nucleus and RNA translation and/or degradation in the cytoplasm, is upregulated by MYC. MYC regulates the expression of many genes involved the transport or shuttling of various RNA species. This coordinated regulation of RNA transport by MYC may directly and indirectly promote the maturation and translation of mRNA and contribute to the maintenance of the cancer phenotype. Thus, these findings highlight RNA transport as a novel genetic vulnerability of MYC-driven cancers and provide a missing piece as to the role of RNA metabolism MYC-driven tumorigenesis. In particular, it is shown that, inhibition of Xpo1, a pivotal receptor in the RNA transport pathway, blocks RNA export, and exhibits marked in vivo therapeutic effects in cancers overexpressing MYC. Xpo1 as a therapeutic target specifically for the treatment of human cancers overexpressing MYC warrants future clinical trials.

REFERENCES