WO2011020012A1 - Lack of expression of tnfa and type 1 receptor for tnfa protects cancer cells from tnfa-induced programmed cell death (cell apoptosis) - Google Patents

Abstract

The present invention includes compositions and methods for determining whether a subject is a candidate for TNFα treatment. The invention includes assessing the methylation status of the TNFα promoter and expression of type 1 TNFα receptor (TNF-Rl) in a cell from the subject. Hypermethylation of the TNFα promoter and/or expression of TNF-Rl is an indication that the subject is suffering from cancer and is capable of responding to TNFα treatment accomplished by induction of expression of endogeneous TNFα and/or administration of exogeneous TNFα.

Description

TITLE OF THE INVENTION

Lack of expression of TNFα and type 1 receptor for TNFα protects cancer cells from

TNFα-induced programmed cell death (cell apoptosis)

BACKGROUND OF THE INVENTION

Although anaplastic large T-cell lymphomas (TCL) share a number of morphologic and immunophenotypic features including almost universal expression of the CD30 antigen, they are clinically and biologically quite heterogeneous (Kadin et al., 2003, Semin Hematol 40: 244-256; Webb et al., 2009, Expert Rev Anticancer

Ther 9:331-356). These lymphomas arise either de novo or constitute a progression of underlying more indolent lymphomas or even pre-malignant lymphoproliferations. TCL expressing anaplastic lymphoma kinase (ALK+ TCL) has been recognized as a distinct entity. This primary systemic lymphoma typically occurs in children and young adults. In contrast, systemic anaplastic large TCL that do not express the kinase typically occur in older subjects and likely represent a pathogenetically diverse group of lymphomas. There is a group of primary CD30+ cutaneous

lymphoproliferative T-cell disorders ranging from lymphomatoid papulosis to an overt anaplastic large T-cell lymphoma, the latter typically arising de novo but sometimes reflecting progression of the former.

TNFα is a pleiotropic cytokine that, among other functions, induces apoptotic cell death (Abe et al., 2003, Blood 101 :1477-1483; van Horssen et al., 2006, Oncologist. 11 :397-408). Diverse extracellular stimuli induce the synthesis of TNFα in a wide variety of cell types, chiefly activated macrophages, T lymphocytes, and natural killer cells. TNFα induces cell apoptosis by cross-linking the type 1 TNFα receptor (TNF-Rl). The cross-linked TNF-Rl activates the signal transduction complex containing a protein called TNFR-associated death domain (TRADD).

The activated TRADD dissociates from the receptor and binds another adaptor protein, Fas-associated death domain (FADD), capable of activating an initiator caspase 8, which in turn triggers effector caspases including caspase 3.

Transcriptional gene silencing due to the DNA methylation of the gene promoter is a common epigenetic event in lymphoid and other malignancies (Jones et al., 2007, Cell 128:683-692; van der Maarel, 2008, Ann Rheum Dis. 67 Suppl 3:iii97-

PHIP/ 832722.1 1 100). It affects diverse tumor suppressor genes, protein products of which affect cell cycle progression, signal transduction, DNA repair, oncogene expression, and other key features of neoplastic cells (Wasik et al., 2009, Semin Oncol 36 (2 Suppl 1):S27- S35). The silencing is mediated by members of the DNA methyltransferase family (DNMT), which induce and maintain methylation of the CpG dinucleotides within promoters of the affected genes. Gene silencing can be reversed by DNMT inhibitors such as 5'-aza-2'-deoxycytidine (5-ADC), and is typically associated with the re- expression of tumor suppressor protein(s) and, consequently, the impaired growth of cancerous cells.

There have been many attempts made to treat cancer, such as TCL.

However, there still exists a need in the art for the development of successful therapies for cancer, including TCL. The present invention satisfies the need in the art for development of new approaches for efficient means to treat cancer including, TCL.

BRIEF SUMMARY OF THE INVENTION

The invention provides compositions and methods for identifying markers of response to therapy of cancer in a mammal. Preferably, the mammal is a human.

In one embodiment, the method comprises detecting a marker in a biological sample derived from a mammal, wherein the marker is selected from the group consisting of hypermethylation of the TNFα promoter compared to the methylation state of TNFα promoter from a biological sample derived from an otherwise normal mammal, present or increased expression of type 1 TNFα receptor (TNF-Rl) compared to the expression of TNF-Rl from a biological sample derived from an otherwise normal mammal, and any combination thereof, further wherein detection of the marker identifies a marker of response to therapy of cancer in the mammal.

In one embodiment, the cancer is lymphoma.

In another embodiment, the biological sample is selected from the group consisting of a tumor tissue or a bodily fluid. In another embodiment, the bodily fluid is peripheral blood.

PHIP/ 832722 1 In one embodiment, the therapy includes, but is not limited to inducing expression of TNF α through reactivation of the TNFα epigenetically silenced gene, inducing expression of exogenous TNFα, administration of exogenous TNFα cytokine, and the like.

The invention also provides a method of diagnosing cancer in a patient.

The method comprises detecting a cancer marker in a biological sample derived from the patient, wherein the marker is selected from the group consisting of

hypermethylation of the TNFα promoter compared to the methylation state of TNFα promoter from a biological sample derived from an otherwise normal patient, present or increased expression of type 1 TNFα receptor (TNF-Rl) compared to the expression of TNF-Rl from a biological sample derived from an otherwise normal patient, and any combination thereof. Detection of the cancer marker is an indication that the patient has cancer.

The invention provides a method of managing treatment of a cancer patient. The method comprises detecting a cancer marker from a biological sample derived from the patient, wherein the marker is selected from the group consisting of hypermethylation of the TNFα promoter compared to the methylation state of TNFα promoter from a biological sample derived from an otherwise normal patient, present or increased expression of type 1 TNFα receptor (TNF-Rl) compared to the expression of TNF-Rl from a biological sample derived from an otherwise normal patient, and any combination thereof. Detection of the cancer marker is an indication that the patient is capable of responding to TNFα therapy.

The invention provides a method for identifying a cell that exhibits unregulated growth. The method comprises measuring the methylation status of a gene in a test cell and a control cell, wherein the control cell exhibits regulated growth; and comparing the methylation status of the gene from the test cell and the control cell, wherein a hypermethylation status of the gene in the test cell compared to the control cell is an indication that the test cell exhibits unregulated growth.

The invention provides a method for identifying a cell that is predisposed to unregulated growth. The method comprises measuring the

methylation status of a gene in a test cell and a control cell, wherein the control cell exhibits regulated growth; and comparing the methylation status of the gene from the test cell and the control cell, wherein a hypermethylation status of the gene in the test

PHIP/ 832722 1 ϊ cell compared to the control cell is an indication that the test cell is predisposed to unregulated growth.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1, comprising Figures IA and IB, is a series of images demonstrating gene expression pattern of the TNFα apoptotic pathway in ALK+ TCL cells. Figure IA depicts the presence (red bar) or absence (blue bar) of expression of the TNFα apoptotic pathway genes detected in the ALK+ TCL-derived SUDHL-I cell line determined by genome-scale DNA oligonucleotide array. Figure IB is a chart depicting expression of the TNFα gene determined by the DNA oligonucleotide array in the SUDHL-I cells cultured in the presence of medium or 5 -ADC for 24, 48, or 72 hours.

Figure 2, comprising Figures 2A and 2B, is a series of images depicting lack of TNFα expression in ALK+ TCL cells. The depicted six ALK+ TCL cell lines as well as two ALK- TCL cell lines (2A and 2B) derived from the CD30+ cutaneous lymphoproliferative T-cell disorder were examined for expression of TNFα mRNA by standard RT-PCR (Figure 2A) and TNFα protein by EIA (Figure 2B). Mitogen(PHA)-stimulated normal PBMC served as a positive control in the protein detection assay.

Figure 3, comprising Figures 3 A through 3C, is a series of images depicting methylation status of TNFα gene promoter in ALK+ TCL cells. Figure 3 A is a schematic map of the CpG sites in the TNFα gene promoter. Figures 3 B and 3 C depict methylation status of TNFα gene promoter in ALK+ TCL and control ALK- TCL cell lines (Figure 3B) and ALK+ TCL tissues (Figure 3C) determined by pyrosequence analysis of the bisulfate-converted DNA.

Figure 4, comprising Figures 4 A through 4C depict the effect of DNA methyltransferase inhibitor 5-ADC on expression of TNFα gene in ALK+ TCL cells. Figure 4A depicts changes in TNFα promoter methylation induced in SUDHL-I cells by treatment with 5-ADC for 0, 24, and 72 hrs. Figure 4B shows changes in TNFα mRNA expression in SUDHL-I cells exposed to 5-ADC for the depicted periods of

PHIP/ 832722 1 A time. Upper panel: standard RT-PCR, lower panels: quantitative RTPCR with GAPDH expression serving as a positive control. Figure 4C depicts the kinetics of 5- ADC induced changes in TNFα protein expression in SUDHL-I (left panel) and Karpas 299 (right panel) detected by EIA.

Figure 5, comprising Figures 5 A and 5B, is a series of images depicting the impact of DNA methylation on activity of TNFα gene promoter. Figure 5A depicts patterns of DNA methylation introduced into the TNFα promoter (open ovals depict unmethylated and solid ovals methylated CpG sites). Figure 5B depicts the corresponding impact of the complete methylation or hemimethylation of proximal or distal TNFα promoter on the promoter's activity as detected by the

Luciferase gene reporter assay. Activity of the unmethylated TNFα promoter examined in parallel, served as a reference.

Figure 6, comprising Figures 6 A through 6D is a series of images depicting TNFα-induced apoptotic cell death of ALK+ TCL cells. Figure 6 A shows the impact of TNFα used at the depicted concentrations on growth of the listed ALK+ and ALK- TCL cell lines as determined in the MTT enzymatic conversion assay. Figure 6B shows the effects of TNFα on caspase 8 and caspase 3 activation status in the depicted ALK+ and ALK- TCL cell lines. Expression of TNF-Rl mRNA (Figure 6C) and protein (Figure 6D) by the depicted six ALK+ and two ALK- TCL cell lines determined by standard RT-PCR and flow cytometry, respectively. In Figure 6D, the orange color line reflects staining with an anti-TNF-Rl antibody, the violet color line is an isotype-matched control antibody.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for identifying markers of response to therapy of cancer in a mammal. Preferably, the mammal is a human. The invention is based on the discovery that form of cancer, the T-cell lymphoma characterized by the expression of anaplastic lymphoma kinase(ALK+ TCL) fails to express TNFα. In some instances, the lack of TNFα expression is associated with at least partial DNA methylation of its gene promoter.

The invention is also based on the discovery that ALK+ TCL cell lines express the type 1 TNFα receptor (TNF-Rl). Thus, the invention includes methods of assessing the methylation status of the TNFα promoter and/or expression of TNF-Rl

PHIP/ 8₃2722.1 as a diagnostic tool to determine whether a patient is a candidate to receive TNFα therapy. A hypermethylation status of the TNFα promoter, detectable expression of TNF-Rl, or the combination thereof is predictive that the patient will be responsive to TNFα therapy. The diagnostic test to determine responsiveness to TNFα therapy is applicable to not only lymphomas, but is applicable to all cancers.

The invention provides compositions and methods of exposing cancer cells including ALK+ TCL cells to TNFα. Exposure to TNFα in induces in cancer cells programmed cell death (cellular apoptosis). In one aspect, apoptosis can be induced in ALK+ TCL cells by treating the cells with a DNA methyltransferase inhibitor. Alternatively, apoptosis can be induced by way of exposing cancer cells to the exogenous TNFα. Regardless of the method of exposing cancer cells to TNFα, inhibition of cell growth corresponds to the induction of members of the cellular apoptotic pathway, such as caspase 8 and caspase 3.

The invention also provides compositions and method of using a DNA methyltransferase inhibitor as a therapy for cancer including ALK+ TCL. The DNA methyltransferase inhibitor serves to reactive the TJSfFa epigenetically silenced gene. In one aspect, the DNA methyltransferase inhibitor is administered to a mammal suffering from cancer including ALK+ TCL.

The invention provides compositions and methods of using TlSfFa as a therapy for cancer. In one aspect, TNFα is administered to a mammal suffering from cancer such as ALK+ TCL.

The invention provides a diagnostic test for distinguishing diseases or conditions that that are associated with hypermethylation of the TNFα promoter from normal methylation of the TNFα promoter. The invention also provides a diagnostic test for distinguishing diseases or conditions that are TNF-Rl positive from the ones that are TNF-Rl negative. The invention also provides a diagnostic test for distinguishing diseases or conditions that are TNF-Rl positive and exhibit hypermethylation of the TNFα promoter from diseases or conditions that are TNF-Rl negative and exhibit normal methylation of the TNFα promoter.

The invention also provides methods for predicting a cancer patient's suitability for treatment with TNFα. The therapy for diseases or conditions that are TISfF-Rl positive and/or exhibit hypermethylation of the TNFα promoter includes

PHIP/ 832722.1 inducing expression of TNFα through reactivation of the TNFα epigenetically silenced gene and/or administering exogenous TNFα to the patient.

Definitions

As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "about" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used.

"Allogeneic" refers to a graft derived from a different individual of the same species.

"Alloantigen" is an antigen that differs from an antigen expressed by the recipient.

The term "ALK" includes the human ALK protein encoded by the ALK (Anaplastic Lymphoma Kinase) gene which in its native form is a membrane- spanning protein tyrosine kinase (PTK)/receptor.

As used herein, the term "autologous" is meant to refer to any material derived from the same individual which it is later to be re-introduced into the individual.

The term "cancer" as used herein is defined as disease characterized by the uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.

The term "DNA" as used herein is defined as deoxyribonucleic acid.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.

PHIP/ 832722 1 Thus, a gene encodes a protein if transcription and translation of mRNA

corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

The term "expression vector" as used herein refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, siRNA, ribozymes, and the like. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

"Liposome", as used herein is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers.

However, the present invention also encompasses compositions that have different structures in solution than the normal vesicular structure. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

PHIP/ 832722.1 8 The term "heterologous" as used herein is defined as DNA or RNA sequences or proteins that are derived from the different species.

"Homologous" as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules.

When a subunit position in both of the two molecules is occupied by the same mononieric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50%

homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 5'- ATTGCC-3' and 5'-TATGGC-3' share 50% homology.

As used herein, "homology" is used synonymously with "identity."

An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

The term "epigenetically silenced" or "epigenetic silencing", when used in reference to a gene, means that the gene is not being transcribed, or is being

PHIP/ 832722. ) Q transcribed at a level that is decreased with respect to the level of transcription of the gene in a corresponding control cell (e.g., a normal cell), due to a mechanism other than a genetic change. Epigenetic mechanisms of gene silencing are well known and include, for example, hypermethylation of CpG dinucleotides in a CpG island of the 5' regulatory region of a gene, and structural changes in chromatin due, for example, to histone acetylation, such that gene transcription is reduced or inhibited. Methods for detecting epigenetic silencing of a gene are disclosed herein and include, for example, detecting re-expression (reactivation) of the gene following contact of a cell with an agent that relieves the epigenetic silencing, for example, with a demethylating agent (e.g., DNA methyltransferase inhibitor) where the silencing is due to

hypermethylation.

As used herein, the term "methylation" or "hypermethylation", when used in reference to a gene, means that cytosine residues of CpG dinucleotides are methylated at the 5'-position, i.e., 5'-methylcytosine. The term "methylation status" is used herein to refer to a relative abundance, including the presence or absence, of methylated cytosine residues of CpG dinucleotides in a CpG island. In general, the cytosine residues in a CpG sites are not methylated in a transcriptionally active gene and, therefore, the detection of methylated cytosine residues in a CpG sites indicates that expression of the gene is reduced or inhibited. Accordingly, as discussed above, reference herein to a "epigenetically silenced" gene means that the gene is not being transcribed, or is being transcribed at a level that is decreased with respect to the level of transcription of the gene in a corresponding control cell (generally a normal cell) due to hypermethylation of CpG dinucleotides in regulatory regions of the gene. A consequence of epigenetically silenced gene expression is that a cell containing the gene has reduced levels of, or completely lacks, a polypeptide encoded by the gene

(i.e., the gene product) such that any function normally attributed to the gene product in the cell is reduced or absent.

As used herein, the term "modulate" is meant to refer to any change in biological state, i.e. increasing, decreasing, and the like. For example, the term "modulate" refers to the ability to regulate positively or negatively the expression of

TNFα, including but not limited to transcription of TNFα mRNA, stability of TNFα mRNA, translation of TNFα mRNA, stability of TNFα polypeptide, TNFα post- translational modifications, or any combination thereof. The term "modulate" also

PHIP/ 832722.1 I Q refers to the ability to regulate positively or negatively the expression of TNF-Rl, including but not limited to transcription of TNF-Rl mRNA, stability of TNF-Rl mRNA, translation of TNF-Rl mRNA, stability of TNF-Rl polypeptide, TNF-Rl post-translational modifications, or any combination thereof.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

The term "polypeptide" as used herein is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term

polypeptide is mutually inclusive of the terms "peptide" and "protein".

As used herein, "predisposition" refers to an increased likelihood that an individual will have a disorder. Although a subject with a predisposition does not yet have the disorder, there exists an increased propensity to the disease.

"Proliferation" is used herein to refer to the reproduction or multiplication of similar forms of entities, for example proliferation of a cell. That is, proliferation encompasses production of a greater number of cells, and can be measured by, among other things, simply counting the numbers of cells, measuring incorporation of ^H-thymidine into the cell, and the like.

The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

PHlP/ 832722 1 1 1 As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements, which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one that expresses the gene product in a tissue specific manner.

A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

The term "RNA" as used herein is defined as ribonucleic acid.

The term "recombinant DNA" as used herein is defined as DNA produced by joining pieces of DNA from different sources.

The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by using recombinant DNA methods.

As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are culture in vitro. In other embodiments, the cells are not cultured in vitro.

"Therapeutically effective amount" is an amount of a compound of the invention, that when administered to a subject, ameliorates a symptom of the disease.

PHIP/ 832722 1 \ 2 The amount of a compound of the invention which constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the age of the subject to be treated, and the like. The therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

"Subject" for the purposes of the present invention includes humans and other animals, particularly mammals, and other organisms. Thus the methods are applicable to both human therapy and veterinary applications. In a preferred embodiment the subject is a mammal, and in a most preferred embodiment the subject is human. The term "subject" is used synonymously herein with the term "patient".

The terms "treat," "treating," and "treatment," refer to therapeutic or preventative measures described herein. The methods of "treatment" employ administration to a subject, in need of such treatment, a composition of the present invention, for example, a subject having a disorder characterized by hypermethylated TNFα promoter region and/or detectable expression of TNF-Rl or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

The term "transfected" or "transformed" or "transduced" as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

The phrase "under transcriptional control" or "operatively linked" as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating

PHIP/ 832722.1 13 plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

"Xenogeneic" refers to a graft derived from an animal of a different species.

Description

The present invention relates to methods and compositions useful for diagnosing and monitoring the progression of cancer. The invention is based on the discovery of a new marker associated with cancer, such as ALK+ TCL and its progression.

The invention relates to the discovery that TNFα gene promoter is hypermethylated and expression of TNFα is undetectable or absent in anaplastic lymphoma cell lines, particularly ALK+ TCL cells. In addition, it was observed that the anaplastic lymphoma cell lines express type 1 TNFα receptor (TNF-Rl). The absence of TNFα leads to an absence of apoptosis, which thereby allows the cancer cells to survive. Adding TNFα to the cells or removal of hypermethylation of the TNFα promoter in the cells to induce expression of the endogeneous TNFα restores apoptosis. Thus, the invention provides compositions and methods for diagnosing cancer in a mammal and determining whether the mammal is a candidate to receive TNFα therapy. A candidate for TNFα therapy exhibits a hypermethylation status of the TNFα promoter and/or expression of TNF-Rl . A hypermethylation status of the TNFα promoter, detectable expression of TNF-Rl , or the combination thereof is predictive that the patient will be responsive to TNFα therapy.

The invention also includes TNFα as a type of therapy for ALK+ TCL through administration of the cytokine to a mammal in need thereof. Alternatively, reactivation of epigenetically silenced TNFα promoter is a therapy for ALK+ TCL through administration of a DNA methyltransferase inhibitor.

The invention is based on the discovery that induced expression of TNFα or administration of exogenous TNFα can be a type of therapy for ALK+ TCL. The present invention relates to the discovery that expression of TNFα by either

PHIP/ 832722 I 14 activating the promoter of the TNFα gene or administration of exogenous TNFα provides a therapeutic benefit for ALK+ TCL. However, the invention should not be construed to be limited to only ALK+ TCL. Rather, the invention encompasses treating any disease or condition associated with hypermethylation of the TNFα promoter and/or undetectable TNFα expression and/or expression of the TNF-Rl .

That is, adding exogenous TNFα to the cells or removal of hypermethylation of TNFα promoter region in the cells restores apoptosis.

Accordingly, the invention includes a method of diagnosing cancer, such as lymphoma, or monitoring the progression of lymphoma in a subject. The method comprises, in a test sample, determining CpG methylation level of the TNFα gene regulatory regions, expression levels of TNFα wherein a statistically significant decrease in the expression of TNFα or non-detectable levels of TNFα indicates the presence of lymphoma or progression of lymphoma to a more invasive form in the subject. Preferably, the lymphoma is ALK+ TCL.

In one embodiment, the invention includes a method of diagnosing cancer, such as lymphoma, or monitoring the progression of lymphoma in a subject. The method comprises, in a test sample, determining expression levels of TNF-Rl wherein expression of TNF-Rl or a statistically significant increase in the expression of TNF-Rl levels indicates the presence of lymphoma or progression of lymphoma to a more invasive form in the subject.

In yet another embodiment, the invention includes a method of diagnosing cancer, such as lymphoma, or monitoring the progression of lymphoma in a subject. The method comprises in a test sample, determining both the expression levels of TNFα and TNF-Rl wherein a statistically significant decrease in the expression of TNFα or non-detectable levels of TNFα indicates the presence of lymphoma or progression of lymphoma to a more invasive form in the subject, further wherein expression of TNF-Rl or a statistically significant increase in the expression of TNF-Rl levels indicates the presence of lymphoma or progression of lymphoma to a more invasive form in the subject.

The invention also includes methods to evaluate, diagnose, and manage treatment of cancer, particularly by detecting or measuring selected molecular cancer markers to provide highly specific, cancer prognostic and/or treatment-related information, and to diagnose and manage pre-cancerous conditions, cancer

PHIP/ 832722.1 \ ζ susceptibility, and cancer prognosis. Preferably, the cancer markers include: 1) statistically significant decrease in the expression of TNFα or non-detectable levels of TNFα; 2) a statistically significant increase in the expression of TNF-Rl levels or detectable levels of TNF-Rl; or 3) the combination thereof.

Accordingly, the analysis of TNFα gene promoter methylation and

TNF-Rl expression is useful in predicting a patient's response to TNFα therapy. For example, a patient that shows hypermethylation of the TNFα promoter, expression of TNF-Rl, or a combination thereof is a candidate for receiving TNFα treatment.

Based on the disclosure presented herein, the analysis for determining whether a patient should receive TNFα treatment is applicable to not only in the context of T- cell lymphoma but all types of lymphoma and, cancer in general.

Epigenetically silenced genes

Although cancers generally are considered to arise from genetic changes such as mutations in a gene, the disclosure presented herein demonstrates that epigenetic mechanisms, which do not result in mutations of the DNA sequence, also can give rise to cancers. The most commonly observed epigenetic change involves silencing of gene expression due to methylation of the gene sequence, particularly the 5' upstream gene regulatory sequences. Methylation of cytosine residues located 5 ' to guanosine in CpG dinucleotides, particularly in CpG-rich regions (CpG islands), often is involved in the normal regulation of gene expression in higher eukaryotes. For example, extensive methylation of CpG islands is associated with transcriptional inactivation of selected imprinted genes, as well as the genes on the inactivated X chromosome in females. Aberrant methylation of normally unmethylated CpG islands also has been found in immortalized and transformed cells, and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers.

The disclosure presented herein demonstrates that TNFα is a tumor suppressor gene in at least TCL, such as ALK+ TCL. This is because it has been discovered that ALK+ TCL cells fail to express TNFα, and restoring expression of

TNFα inhibits their growth by inducing cellular apoptosis.

As such, screening assays directed to detecting the methylation status of the TNFα promoter can provide diagnostic information relating to cancer. As

PHIP/ 832722 1 \β cancer often is a silent disease that does not present clinical signs or symptoms until the disease is well advanced, the availability and use of the methylation status of the TNFα promoter as a marker allows the identification of individuals that are susceptible to a cancer. As discussed elsewhere herein, in some instances, the screening assays of the invention also include detecting the level of TNF-Rl in combination with measuring the methylation status of the TNFα promoter as a marker for cancer.

The present invention relates to methods of identifying epigenetically silenced genes, for example, methylation silenced genes, that are associated with a cancer. In one embodiment the present invention relates to a method of identifying at least one epigenetically silenced gene associated with at least one cancer. Such a method can be performed, for example, by contacting an array of nucleotide sequences representative of a genome with nucleic acid subtraction products under conditions suitable for selective hybridization of nucleic acid subtraction products to complementary nucleotide sequences of the array, and detecting selective

hybridization of nucleic acid subtraction products to a subpopulation of nucleotide sequences of the array. Preferably, the nucleic acid subtraction products comprise nucleic acid molecules corresponding to RNA expressed in cancer cells contacted with at least one agent that reactivates expression of epigenetically silenced genes but not RNA expressed in normal cells corresponding to the cancer cells. Identification of at least one epigenetically silenced genes associated with at least one cancer is accomplished when the nucleic acid molecules corresponding to RNA expressed in the normal cells corresponding the cancer cells do not hybridize to the subpopulation of nucleotide sequences under such conditions suitable for selective hybridization because the nucleic acid subtraction products that selectively hybridize to the subpopulation of nucleotide sequences of the array represent epigenetically silenced genes of the cancer cells.

In one embodiment, the epigenetically silence gene is TNFα. In another embodiment, the epigenetically silenced TNFα gene is associated with ALK+ TCL. Accordingly, a therapeutic agent to treat a disease or condition associated with epigenetically silenced TNFα gene is an agent that can reactivate expression of epigenetically silenced genes. Any such agent, for example, a methyltransferase inhibitor (e.g., 5-aza-2'-deoxycytidine; DAC), a histone deacetylase inhibitor (e.g.,

PHIP/ 832722.1 \ η trichostatin A; TSA), or a combination of agents such as a combination of DAC and TSA is encompassed by the invention.

The present invention provides a method for identifying a cell that exhibits or is predisposed to exhibiting unregulated growth by detecting, in a test cell, epigenetic silencing of the TNFα gene.

In one embodiment, a method of the invention includes a comparison of the methylation status of a gene in a test cell or sample with the methylation status of a corresponding gene in a corresponding cell exhibiting regulated growth. The method includes measuring both the methylation status of a gene in a test cell or sample and the methylation status of the corresponding gene in a corresponding cell exhibiting regulated growth, and comparing the methylation status of the gene from the samples. Hypermethylation of the gene is associated with a cancer diagnosis. Hypermethylation is a well known term in the art which denotes increased and aberrant methylation at specific CpG sites in a gene. Hypermethylation most often occurs in the promoter regions of genes and acts to depress expression of the gene.

Accordingly, in a preferred embodiment, the method is carried out wherein the promoter regions of the relevant genes are assessed to determine their methylation status, in particular whether the promoter region is hypermethylated.

As used herein, the term "corresponding" means a reference material, with which a test material is being compared. Generally, the reference material provides a control or standard with which the test material is compared. For example, reference to a corresponding unmethylated TNFα gene, with respect to an TNFα gene being examined for methylation status, means that the unmethylated TNFα gene is the same type of gene as the TNFα gene being examined for methylation status, e.g., the test gene and the corresponding unmethylated gene are both human TNFα genes.

Reference to a corresponding cell exhibiting regulated growth, with respect to a test cell, generally refers to a normal cell, i.e., a cell that has a cell cycle and growth pattern characteristic of a population of such cells in a healthy individual.

The present invention also relates to a method of reducing or inhibiting unregulated growth of a cell exhibiting epigenetic silenced transcription of at least one gene associated with a cancer. The method comprises reactivating the promoter region of the silenced gene in the cell, thereby restoring promoter activity of the promoter region of the gene. Reactivation of the promoter region serves to increase

PHIP/ 832722 1 ^ g transcription of the gene and expression of the polypeptide encoded by the epigenetic silenced gene in the cell. In one aspect, expression of a gene and associated polypeptide can be restored, for example, by contacting the cell with a demethylating agent (e.g., a DNA methyltransferase inhibitor), a histone deacetylase inhibitor, or a combination thereof.

In one embodiment, the invention comprises a composition for removing hypermethylation of the TNF α promoter region.

In one embodiment, the epigenetic silenced gene is a methylation silenced gene, and the method includes contacting the cell with at least one DNA methyltransferase inhibitor, for example, 5-ADC. In one aspect, the cell can be contacted with the methyltransferase inhibitor in vitro, e.g., in a culture medium or other medium conducive to survival of the cell. If desired, the cell contacted with the methyltransferase inhibitor further can be administered to a subject. In another aspect, the agent can be administered to the subject such that the cell exhibiting unregulated growth is contacted with the agent.

The present invention further relates to a method for treating a cancer subject, wherein cancer cells in the subject exhibit epigenetic silenced expression of at least one gene. Such a method can be performed, for example, by restoring expression of one or more epigenetic silenced genes in cancer cells in the subject. The method comprises reactivating the promoter region of the silenced gene in the cell, thereby restoring promoter activity of the promoter region of the gene.

Reactivation of the promoter region serves to increase transcription of the gene and expression of the polypeptide encoded by the epigenetic silenced gene in the cell. In one aspect, expression of a gene and associated polypeptide can be restored, for example, by administering a methyltransferase inhibitor to the subject in an amount sufficient to restore expression of the methylation silenced gene(s) in cancer cells in the subject, thereby treating cancer in the subject.

The present invention also relates to a method for selecting a therapeutic strategy for treating a cancer subject. Such a method can be performed, for example, by identifying at least one methylation silenced gene associated with the cancer, according to a method as disclosed elsewhere herein. For example, when a cell or biological sample derived from the subject is identified to have at least one epigenetically silenced gene associated with at least one cancer, the therapeutic

PHIP/ 832722 1 19 strategy for treating the subject can include restoring expression of the identified epigenetic silenced gene. Restoring the expression of the epigenetic silenced gene can be accomplished by administering a DNA methyltransferase inhibitor to the subject in an amount sufficient to restore expression of the silenced gene in cancer cells in the subject, thereby treating cancer in the subject. An alternative strategy can include administering TNFα to the subject.

Expression of one or more methylation silenced genes such as TNFα gene can be restored, for example, by contacting the cells with a DNA

methytransferase inhibitor such as 5-ADC, which, when incorporated into the genes during replication of the cell results in progeny cells containing unmethylated genes, which can be transcribed. The cells contacted with the methytransferase inhibitor can be cells in culture, wherein the methytransferase inhibitor is added to the cell culture medium in an amount sufficient to result in demethylation of the target genes, without being toxic to the cells. The cells in culture can be cells of an established cell line, or can be cells, which can be a mixed population of cells, that have been removed from a subject and are being contacted ex vivo, for example, to determine whether contact with the particular methytransferase inhibitor can restore expression of the target gene(s), and therefore, can be useful when administered to the subject. Such ex vivo treatment of the cells also can be useful for restoring expression of the target gene, after which the cells, which optionally can be expanded in culture, can be

administered back to the subject. Such a method, as well as any of the methods of treatment as disclosed herein, can further include treatments otherwise known in the art as useful for treating a subject having the particular cancer, or that can be newly useful when used in combination with the present methods.

Cells exhibiting methylation silenced gene expression also can be contacted with a methytransferase inhibitor in vivo by administering the agent to a subject. Where convenient, the methytransferase inhibitor can be administered using, for example, a catheterization procedure, at or near the site of the cells exhibiting unregulated growth in the subject, or into a blood vessel in which the blood is flowing to the site of the cells. Similarly, where an organ, or portion thereof, to be treated can be isolated by a shunt procedure, the agent can be administered via the shunt, thus substantially providing the agent to the site containing the cells. The agent also can

PHIP/ 832722.1 20 be administered systemically or via other routes as disclosed herein or otherwise known in the art.

Protein therapy

The present invention relates to a method of diagnosing a subject suffering from a cancer such as TCL, wherein cells associated with the cancer contain at least one methylation silenced gene and/or expression of TNF-Rl . Preferably, the silence gene is TNFα. Diagnosing a subject for methylated silenced TNFα, expression of TNF-Rl, or a combination thereof is useful in predicting the subject's response to TNFα therapy.

Diagnosing a subject suffering from a cancer associated with a methylated silenced gene is discussed elsewhere herein. Briefly, the diagnostic method includes measuring the methylation status of a gene in a cell or sample derived from the subject and measuring the methylation status of the corresponding gene in a corresponding cell derived from an otherwise healthy subject, and comparing the methylation status of the gene from the samples. Hypermethylation of the gene is associated with a cancer diagnosis.

With respect to measuring the expression level of TNF-Rl, any means known in the art can be used. Either mRNA or protein can be measured. Methods employing hybridization to nucleic acid probes can be employed for measuring specific mRNAs. Such methods include using nucleic acid probe arrays (microarray technology) and using Northern blots. Messenger RJVA can also be assessed using amplification techniques, such as RT-PCR. Advances in genomic technologies permit the simultaneous analysis of thousands of genes, although many are based on the same concept of specific probe-target hybridization. Sequencing-based methods are an alternative; these methods started with the use of expressed sequence tags (ESTs), and now include methods based on short tags, such as serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS). Differential display techniques provide yet another means of analyzing gene expression; this family of techniques is based on random amplification of cDNA fragments generated by restriction digestion, and bands that differ between two tissues identify cDNAs of interest. Specific proteins can be assessed using any convenient method including immunoassays and immuno-cytochemistry but are not limited to these specific

PHIP/ 832722 1 21 methods. Most such methods will employ antibodies which are specific for the particular protein or protein fragments. However, the invention is not limited to any particular method of measuring the level of TNF-Rl.

In one embodiment, the levels of gene expression are determined using RT-PCR. Reverse transcriptase polymerase chain reaction is a well-known technique in the art which relies upon the enzyme reverse transcriptase to reverse transcribe mRNA to form cDNA, which can then be amplified in a standard PCR reaction.

Protocols and kits for carrying out RT-PCR are extremely well known to those of skill in the art and are commercially available.

By identifying a novel marker linked to cancer, such as lymphoma, new treatments for this disease may be uncovered. The novel marker which includes epigenetically silenced TNFα, expression of TNF-Rl, or a combination thereof, is useful in predicting the subject's response to TNFα therapy. Thus, the expression pattern of TNFα and TNF-Rl linked to cancer and in particular lymphoma may be used as a research tool to identify new pharmaceuticals which may be used to treat, prevent or control cancer.

The present invention also includes a method of treating a subject suffering from a cancer such as lymphoma, wherein cells associated with the cancer contain at least one methylation silenced gene and/or expression of TNF-Rl .

Preferably, the silenced gene is TNFα. Such a method can be performed, for example, by administering an amount of an agent that restores expression of the at least one methylation silenced gene to the subject sufficient to restore expression of the methylation silenced gene in cells associated with the cancer. The agent can be a polynucleotide encoding the at least one methylation silenced gene, for example, a polynucleotide encoding a polypeptide encoded by a TNFα, a family member thereof, or a combination thereof, or can be a methyltransferase inhibitor such as 5-ADC. An agent useful for treating a subject suffering from a cancer, such as ALK+ TLC, can be administer to a site of the cancer cells in the subject.

The subject can be treated by administering at least one polynucleotide encoding at least one polypeptide encoded by one or more of the epigenetic silenced genes to the subject under conditions sufficient for expression of the at least one polypeptide in cancer cells in the subject. Where a polynucleotide is administered to the subject, the polynucleotide can be contained in a vector (e.g., a viral vector)

PHlP/ 832722.1 22 preferably an expression vector, and/or can be formulated in a matrix that facilitates uptake of the polynucleotide by a target cancer cell (e.g., in a liposome).

In another embodiment, the method includes introducing a

polynucleotide encoding the polypeptide into the cell, whereby the polypeptide is expressed from the polynucleotide, thereby restoring expression of the polypeptide in the cell. The polynucleotide can, but need not, be contained in a vector, e.g., a viral vector, and/or can be formulated in a matrix that facilitates introduction of the polynucleotide into a cell, e.g., liposomes or microbubbles. The polynucleotide can be introduced into a cell by contacting the cell with the polynucleotide ex vivo, in which case the cell containing the polynucleotide can, but need not, be administered to a subject. The polynucleotide also can be introduced into a cell by contacting the cell with the polynucleotide in vivo.

As such, the present invention provides methods of gene therapy, which can be practiced in vivo or ex vivo. For example, where the cell is

characterized by epigenetically silenced transcription of the TNFα gene, a

polynucleotide having a nucleotide sequence encoding TNFα protein can be introduced into the target cell or subject.

In other related aspects, the invention includes at least one polynucleotide encoding at least one polypeptide encoded by one or more of the epigenetic silenced genes operably linked to a nucleic acid comprising a

promoter/regulatory sequence such that the nucleic acid is preferably capable of directing expression of the protein encoded by the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2001, Molecular Cloning: A

Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

The desired polynucleotide can be cloned into a number of types of vectors. However, the present invention should not be construed to be limited to any particular vector. Instead, the present invention should be construed to encompass a wide plethora of vectors which are readily available and/or well-known in the art. For example, a desired polynucleotide of the invention can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal

PHIP/ 832722.1 23 virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Numerous expression vector systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-vector based systems can be employed for use with the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.

Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001), and in Ausubel et al. (1997), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193.

For expression of the desired polynucleotide, at least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional promoter elements, i.e., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

PHIP/ 832722 1 24 A promoter may be one naturally associated with a gene or

polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence.

Alternatively, certain advantages will be gained by positioning the coding

polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2001). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

A promoter sequence exemplified in the experimental examples presented herein is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving

PHIP/ 832722.1 25 high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney virus promoter, the avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the muscle creatine promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter in the invention provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

Further, the invention includes the use of a tissue specific promoter, which promoter is active only in a desired tissue. Tissue specific promoters are well known in the art and include, but are not limited to, the HER-2 promoter and the PSA associated promoter sequences.

In order to assess the expression of the desired gene, for example

TNFα, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. Reporter genes that encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that

PHIP/ 832722.1 26 encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.

Suitable reporter genes may include genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (see, e.g., Ui-Tei et al., 2000 FEBS Lett. 479:79- 82). Suitable expression systems are well known and may be prepared using well known techniques or obtained commercially. Internal deletion constructs may be generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. Constructs may then be transfected into cells that display high levels of siRNA polynucleotide and/or polypeptide expression. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical or biological means. It is readily understood that the introduction of the expression vector comprising the polynucleotide of the invention yields a silenced cell with respect to a regulator.

Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example,

Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

PHIP/ 832722 1 27 Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes,

nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

Any DNA vector or delivery vehicle can be utilized to transfer the desired polynucleotide to a cell in vitro or in vivo. In the case where a non-viral delivery system is utilized, a preferred delivery vehicle is a liposome. The above- mentioned delivery systems and protocols therefore can be found in Gene Targeting Protocols, 2ed., pp 1-35 (2002) and Gene Transfer and Expression Protocols, Vol. 7,

Murray ed., pp 81-89 (1991).

Gene Therapy Administration

One skilled in the art recognizes that different methods of delivery may be utilized to administer a vector into a cell. Examples include: (1) methods utilizing physical means, such as electroporation (electricity), a gene gun (physical force) or applying large volumes of a liquid (pressure); and (2) methods wherein the vector is complexed to another entity, such as a liposome, aggregated protein or transporter molecule.

Furthermore, the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism. Similarly, amounts can vary in in vitro applications depending on the particular cell line utilized (e.g., based on the number of vector

PHIP/ 832722 1 28 receptors present on the cell surface, or the ability of the particular vector employed for gene transfer to replicate in that cell line). Furthermore, the amount of vector to be added per cell will likely vary with the length and stability of the therapeutic gene inserted in the vector, as well as also the nature of the sequence, and is particularly a parameter which needs to be determined empirically, and can be altered due to factors not inherent to the methods of the present invention (for instance, the cost associated with synthesis). One skilled in the art can easily make any necessary adjustments in accordance with the exigencies of the particular situation.

Cells containing the therapeutic agent may also contain a suicide gene i.e., a gene which encodes a product that can be used to destroy the cell. In many gene therapy situations, it is desirable to be able to express a gene for therapeutic purposes in a host, cell but also to have the capacity to destroy the host cell at will. The therapeutic agent can be linked to a suicide gene, whose expression is not activated in the absence of an activator compound. When death of the cell in which both the agent and the suicide gene have been introduced is desired, the activator compound is administered to the cell thereby activating expression of the suicide gene and killing the cell. Examples of suicide gene/prodrug combinations which may be used are herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine;

thymidine kinase thymidilate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside.

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

EXAMPLES

The experiments disclosed herein demonstrate that T-cell lymphomas characterized by the expression of anaplastic lymphoma kinase (ALK+ TCL) fail to express the TNFα and frequently display DNA methylation of the TNFα gene promoter. The results indicate that while only a subset of the ALK+ TCL-derived cell lines showed a high degree of the promoter methylation, all six cell lines tested

PHIP/ 832722 1 29 showed low to non-detectable expression of the TNFα mRNA and none expressed the TNFα protein. All fourteen ALK+ TCL tissue samples examined displayed some degree of the TNFα promoter methylation, which was the most prominent in the distal portion of the promoter. Treatment with a DNA methyltransferase inhibitor, 5'- aza-2'-deoxy-cytidine (5 -ADC), reversed the promoter methylation and led to the expression of TNFα mRNA and protein. Furthermore, in vitro DNA methylation of the promoter impaired its transcriptional activity in the luciferase reporter assay. This impairment was observed even if only either distal or proximal portion were methylated, with methylation of the former exerting a more profound inhibitory effect. Notably, the ALK+ TCL cell lines uniformly expressed the type 1 TNFα receptor (TNF-Rl) protein known to transduce the TNFα-induced pro-apoptotic signals. Moreover, exogenous TNFα inhibited growth of the ALK+ TCL cell lines in a dose-dependent manner and induced activation of the members of the cell apoptotic pathway: caspase 8 and caspase 3. These findings provide support for the therapeutic inhibition of DNA methyltransferases in ALK+ TCL. The results presented herein also indicate that treatment with TNFα can be highly effective in lymphomas, particularly ALK+ TCL.

The materials and methods employed in the experiments disclosed herein are now described.

Cells and tissue samples

SUDHL-I, Karpas 299, JB6, SUP-M2, L82, and SR786 cell lines were derived from ALK+ TCL. Two ALK- TCL cell lines (2 A and 2B lines) were developed from the clinically advanced stages of the progressive CD30+ cutaneous lymphoproliferative T-cell disorder. Peripheral blood mononuclear cells (PBMC) harvested from healthy adults were isolated by Ficoll/Paque centrifugation and stimulated in vitro for 72 hrs with a mitogen (PHA; Sigma). ALK+ TCL tissues were from lymph nodes or extranodal tumors as excisional biopsies obtained for diagnostic purposes. The diagnosis was established by standard morphological and immunohistochemical criteria including expression of the CD30 and ALK proteins.

In the DNA isolation step, the snap-frozen, glass slide-deposited tissue sections were enriched for the lymphoma cells by collecting only the section parts containing

PHIP/ 832722.1 30 predominantly the malignant cells as determined by microscopic evaluation of the H&E stained control slides.

Treatment of the ALK+ TCL cells with DNMT inhibitor

The cells were treated with 0.5 μM of 5-ADC (Sigma) for up to 4 days with replenishing the culture medium with a freshly prepared drug after 2 days. The cells were harvested typically at the 24 hr intervals and subjected to DNA, RNA, and protein extraction. DNA oligonucleotide array assay

The total RNA from cells treated in triplicate cultures with 5-ADC was reverse-transcribed, biotin-labeled, and hybridized to the U 133 Plus 2.0 array chips (Affymetrix) containing 54,000 DNA oligonucleotide probe set. The results were normalized using GeneSpring and further analyzed using Partek GS, Spotfire, and GeneSpring programs.

RT-PCR and quantitative RT-PCR

RNeasy kit (Qiagen)-extracted RNA was treated with Superscript II reverse transcriptase (GIBCO/BRL) and purified on Centri-Sep columns (Princeton Separations). PCR was performed with primers specific for TNFα (5'-

CCCCAGGGACCTCTCTCTAA-3'; SEQ ID NO:1 and 5'- TGAGGTACAGGCCCTCTGAT-3'; SEQ ID NO:2), TNF-Rl (5'- TCTATGCCCGAGTCTCAACC-3'; SEQ ID NO:3 and 5'- GGTGAGGGACCAGTCCAATA-3'; SEQ ID NO:4) as well as GAPDH (5'- TCTCCAGAACATCATCCCTGCCTC-3'; SEQ ID NO:5 and 5'-TGGGCC

ATGAGGTCCACCACCCTG-3'; SEQ ID NO:6). The conventional PCR was performed in duplicate for 30 cycles in the standard reaction conditions. The quantitative ("real time") PCR was performed using the LightCycler 480 Real-Time PCR System from Roche. Representative samples were analyzed by agarose gel electrophoresis to confirm the specificity and size of the PCR-amplified product. The data were normalized to expression of the control glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) mRNA. The fold change in specific mRNA concentration was calculated using the comparative CT method. Results are presented as mean ±

PHIP/ 832722.1 3 1 SEM of the fold change calculated from three experiments, each performed in triplicate.

Western blotting

These experiments were performed using ECL chemiluminescence and antibodies against caspase-8 and caspase-3 (Cell Signaling Technology) according to the standard protocols.

DNA methylation analysis

DNA isolated using the DNeasy Tissue Kit (Qiagen) was bisulfite- modified using the CpGenome DNA Modification Kit (Intergen) and amplified by PCR in 40 cycles in standard conditions using TNFα gene promoter specific biotynylated primers (5'-GTTTTTAAA AG AAATGG AGGT AATAGGTT-3¹; SEQ ID NO:7 and S'-TCCCTCTTAACTAATCCTCTACTATCCT-S'; SEQ ID NO:8) and Platinum Taq DNA Polymerase (Invitrogen). The PCR amplification products were purified and rendered single-stranded on a Pyrosequencing workstation

(Pyrosequencing AB) and annealed to the sequencing primers (5'- GGTTTTGAGGGGTATG-3'; SEQ ID N0:9, 5'-GGAGTGTGAGGGGTATT-S' ; SEQ ID NO: 10, and 5 '-GGGTTTTTTTATTAAGGAAGTTT-S '; SEQ ID NO: 11). The quantitative DNA methylation analysis was performed on a PSQ 96MA system with the PyroGold SQA reagent kit (Pyrosequencing), and results were analyzed using the Q-CpG software (V 1.0.9; Pyrosequencing). All primers were designed with the PSQ Assay Design software (Biotage). All samples were tested in duplicate. Luciferase reporter assay

The TNFα promoter DNA sequence was amplified by PCR with primers 5'-GGTACCAAGAAATGGAGGCAATAG-S '; SEQ ID NO: 12 and 5' - CTCGAGCTCTTAGCTGGTCCTCTG-3'; SEQ ID NO: 13 (the underlined sequences represent nucleotides added to the complementary sequences to generate Kpnl - and Xhol -specific restriction digest sites. The 480-bp PCR product was gel purified and cloned into the pGL3 -basic luciferase reporter construct (Promega) to generate the TNFα-promoter-pGL3 construct. The construct was validated by sequence analysis. To generate construct containing all 12 methylated CpG sites

PHIP/ 832722.1 32 present in the TNFα promoter, the insert promoter DNA was digested with Kpnl and Xhol and methylated by Sssl methylase (New England Biolabs), then cloned back to pGL3-basic luciferase reporter construct. To produce the constructs with

partial (hemi-) methylation, TNFα promoter was digested with Kpnl and PspOMI or PspOMI and Xhol as the distal and proximal portions and then the portions were methylated by Sssl methylase respectively. Methylated or unmethylated fragments, were ligated with the core pGL3-basic constructs. The ligated products were run on argrose gel to confirm ligation efficacy. HEK293 cells were next transiently transfected in duplicate with the construct using Lipofectamine 2000 (Invitrogen) and SuperFect Transfection Reagent (Qiagen) according to the manufacturer's directions, with the combination of the luciferase constructs and phRL (renilla luciferase) TK plasmids (Promega), the latter serving as a measure of transfection efficiency.

Twenty-four hours after transfection, the cells were washed, lysed, and sequentially evaluated for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was measured for 10 seconds after a 2-second delay using a BD Monolight 3010 luminometer (BD Biosciences). Variation in transfection efficiency was normalized by dividing the construct luciferase activity by the corresponding renilla luciferase activity. Promoter activity is reported as the mean ± SD.

EIA assay for T]SfFa

TNFa expression was measured using ELISA kits (R&D Systems) according to the manufacturer's protocol using cell culture supernatants.

Recombinant human TNFα was used to establish a standard curve. The results were obtained using a colorimetric plate reader from Bio-Rad. Flow cytometry. The cells were incubated with the PE-conjugated anti-TNF-Rl or control IgG antibody (R&D Systems) for 1 hour at room temperature. After washing in PBS, the cells were incubated in the secondary antibody for 1 hour. Flow cytometry (FACSCalibur; BD Biosciences) analysis is performed with the CellQuest Pro software.

The results of the experiments presented in this Example are now described.

PHlP/ 832722 1 33 Example 1 : ALK+ TCL cells lack expression of TNFα

ALK+ TCL cells lack expression of TNF ALK+ TCL cells lack expression of TNFα. To identify the potential epigenetically-silenced tumor suppressor genes, ALK+ TCL cell line SUDHL-I were examined for changes in response to the DNA methyltransferase inhibitor 5- ADC using a whole-genome

DNA oligonucleotide array-based gene expression profiling approach. It was observed that one of the important cytokines absent in the untreated cells was TNF ALK+ TCL cells lack expression of TNFα. However, the proteins involved in TNFα-induced signaling were all expressed (Figure IA) suggesting a selective loss of TNFα expression. Of note, cell treatment with 5-ADC led to a marked increase in the

T]VFa mRNA expression in a time-dependent manner (Figure IB), while the expression of the other members of the TNFα signaling pathway remained essentially unchanged.

To determine the TNFα expression status in a larger pool of the ALK+ TCL cell populations, six different ALK+ TCL-derived cell lines as well as two ALK-

TCL lines derived from the primary CD30+ cutaneous lymphoproliferative T-cell disorder were examined. While the ALK- TCL cells strongly expressed TNFα mRNA, a weak to non-detectable message was present in the ALK+ TCL cells (Figure 2A). None of the six ALK+ TCL cell lines tested produced the TNFα protein whereas the cytokine was synthesized in great abundance by the ALK- TCL lines as compared to mitogen-stimulated normal mononuclear cells serving as a positive control (Figure 2B).

Example 2: Methylation of the TNFα promoter in ALK+ TCL cells

In view of the fact that the gene expression profiling data (Figure 1 B) strongly suggested that the TNFα gene is epigenetically silenced, at least in the SUDHL-I cell line, the next set of experiments was designed to evaluate the DNA methylation status of the gene's promoter. The TNFα gene promoter contains twelve CpG sites (Figure 3A) and all these sites displayed a high degree of methylation in the SUDH-Ll and another ALK+ TCL cell line, Karpas 299, as determined by the DNA pyrosequencing analysis of bisulfate-modified DNA (Figure 3B). Two ALK+ TCL cell lines examined (SR786 and L82), as well as two ALK- TCL lines showed little or no methylation. None of the two ALK+ TCL showed any deletions or point

PHIP/ 832722 1 34 mutations within the TNFα gene coding sequence, splice junctions, or promoter region.

The next set of experiments was designed to determine the promoter's CpG methylation status in the primary ALK+ TCL using snap frozen tissues samples. Although quantitatively diverse, likely due to differences in the non-malignant cell content, the same methylation pattern was, however, identified in all lymphoma tissue samples (Figure 3C). While the distal portion of the promoter showed a high and gradually decreasing degree of methylation, the proximal part was much less methylated.

Example 3: Methylation of the TNFα gene promoter suppresses transcription of the

To assess the impact of the CpG site methylation on the transcriptional activity of the TNFα gene, ALK+ TCL SUDHL-I cells, which contain a highly methylated TNFα promoter were treated with 5 -ADC and evaluated the cells for changes in the promoter methylation (Figure 3B). Whereas the cell exposure to the drug for 24 hrs had little detectable effect on the promoter's methylation in the DNA pyrosequencing analysis, treatment for 72 hrs profoundly decreased the methylation level (Figure 4A). 5-ADC treatment also induced the expression of TNFα mRNA in a time dependent manner, as determined by both standard semiquantitative and fully-quantitative RT-PCR (Figure 4B). This increase in the mRNA concentration was associated with a parallel increase in synthesis of the TNFα protein, as shown in the SUDHL-I and Karpas 299 ALK+ TCL cell lines

(Figure 4C). The results demonstrate that the promoter methylation indeed prevents the expression of the TNFα gene.

A luciferase reporter assay was used to provide direct evidence that the promoter methylation impairs its functional transcriptional activity. Because previous reports (Chiu et al., 1997, Arterioscler Thromb Vase Biol 17:3570-3577; Hinchliffe et al., 2002, Biochem J 363:563-570; Satoh et al., 2003, Exp Diabesity Res 4:65-71) indicated that methylation of the carrier vector significantly reduces the

transcriptional activity of the inserted gene promoter, reporter constructs in which the TNFα gene promoter underwent methylation prior to being ligated into the vector was prepared. As shown in Figure 5 (upper panel), the promoter methylation has

PHIP/ 832722.1 35 decreased its activity by almost 10-fold as compared to the unmethylated promoter. Considering the preferential methylation of the distal portion of the TNFα gene promoter seen in the ALK+ TCL tissue samples (Figure 3C) and methylation of the entire promoter in a subset of the ALK+ TCL cell lines (Figure 3A), the relative contribution of methylation of the distal and proximal parts of the promoter to the inhibition of its transcriptional activity was examine. As seen in Figure 5, methylation of both, the distal (middle panel) and proximal (lower panel) portions markedly reduced the promoter activity. While methylation of the distal part was almost as potent as methylation of the entire promoter in inhibiting its activity, selective methylation of the proximal region had a somewhat less profound effect.

Example 4: TNFα induces apoptotic cell death of ALK+ TCL cells

Given the universal lack of TNFα protein expression among the ALK+ TCL cell lines (Figure 2B), the effect of recombinant TNFα on their growth was examined (Figure 6A). TNFα induced a strong, concentration-dependent inhibition of the cell line growth as determined by their ability to convert the MTT substrate. Of note, growth of the ALK- TCL cell lines which constitutively produce TNFα (Figure 2B) remained completely unaffected by the exogenous TNFα. Because the MTT conversion assay reflects mainly cell viability, the impact of TNFα on the activation status of two key enzymes, caspase 8 and caspase 3, involved in mediating apoptotic cell death was determined. As shown in Figure 6B, TNFα activated both caspases in a dose-dependent manner as indicated by a steadily reduced concentration of their inactive full-length proenzymatic forms and proportionately increased expression of the cleaved, active products. The caspase activation occurred only in the ALK+ TCL cells (right panel) but not the control ALK- TCL cells (left panel).

Because TNF-Rl has been identified as responsible for transducing the proapoptotic signals of TNFα, the expression status of the receptor in several ALK+ and ALK- TCL cell lines was examined using RT-PCR and flow cytometry. Whereas all six ALK+ TCL cell lines tested strongly expressed TNF-Rl mRNA (Figure 6C) and protein (Fig. 6D), the receptor was not expressed by the two ALK- TCL lines tested. This observation indicates that the differential expression of TNF-Rl is responsible for the difference in apoptotic response to TNFα between these two types of T-cell lymphoma.

PHIP/ 832722 1 36 Example 5: Lack of TNFα expression protects ALK+ TCL cells from apoptosis

The results presented herein demonstrate that that ALK+ TCL cells universally fail to express TNFα despite originating from CD4+ T lymphocytes. TNFα is a major source of the cytokine under physiological conditions (Abe et al.,

2003, Blood 101:1477-1483; van Horssen et al., 2006, Oncologist. 11 :397-408). This lack of expression is typically related to the at least partial DNA methylation of the TNFα gene promoter, as demonstrated by the induction of the TNFα expression by a DNMT inhibitor 5 -ADC, on one hand, and the impairment of the promoter activity by not only the complete but also partial DNA methylation, on the other. In contrast to

TNFα, ALK+ TCL cells uniformly express TNF-Rl and their treatment with the cytokine leads to apoptotic cell death.

Previous studies indicate that concomitant epigenetic silencing of various tumor suppressor genes by DNA methylation comprises the major mechanism of malignant cell transformation in ALK+ TCL and, likely, other malignancies

(Wasik et al., 2009, Semin Oncol 36 (2 Suppl 1):S27-S35). Furthermore, it affects diverse cell functions and programs, persistent activation of which is critical to the malignant cell phenotype. Accordingly, ALK+ TCL cells display silencing of the pl6(INK4a) gene (Nagasawa et al., 2006, Leuk Res 30:303-312) that encodes a key inhibitor of the cell cycle progression at the Gl/S transition phase by suppressing the

CyclinDCDK4/6 complex and, therefore, facilitating uncontrolled growth of the malignant cells. Similar to cutaneous TCL (Zhang et al., 2000, Am J Pathol

157:1137-1146) and other hematopoietic cell malignancies (Oka et al., 2002, Cancer Res 62:6390-6394), ALK+ TCL also show silencing of the SHP-I gene (Khoury et al., 2004, Blood 104: 1580-1581; Honorat et al., 2006, Blood 107:4130-4138; Zhang et al., 2005, Proc Natl Acad Sci USA 102:6948-6953). SHP-I is a tyrosine phosphatase that in normal immune cells negatively regulates signaling through cell- surface receptors for cytokines, chemokines, and antigens (Wu et al., 2003, Gene 306:1-12) by dephosphorylating the receptors, receptor-associated Jak kinases, and other proteins. Forced expression of SHP-I in ALK+ TCL cells inhibits

phosphorylation of NPM/ALK, a hybrid protein form in which ALK is typically expressed in this type of lymphoma. Importantly, the SHP-I -mediated

dephosphorylation inhibits enzymatic activity of NPM/ALK and fosters ubiquitin-

PHIP/ 832722.1 37 dependent degradation of the oncogenic kinase (Honorat et al., 2006, Blood

107:4130-4138). ALK+ TCL cells epigenetically silence the STAT5a gene (Zhang et al., 2007, Nature Med. 13: 1341-1348). Remarkably, expression of the STAT5a protein selectively inhibits expression of the NPM/ALK gene indicating that STAT5a acts in ALK+ TCL cells as a bona fide tumor suppressor gene and its silencing, together with the silencing of the SHP-I gene, is critical for undisturbed expression, and hence cell transforming properties, of ALK (Wasik et al., 2009, Semin Oncol 36 (2 Suppl 1):S27-S35).

The results presented herein demonstrate that ALK+ TCL cells silence the TNFα gene and that TNFα induces apoptosis of the ALK+ TCL cells not only define TNFα gene as a tumor suppressor gene but also add protection from apoptotic cell death as another oncogenic effect of DNA methylation. In addition, the results presented herein indicate that cells derived from the ALK- TCL express TNFα but fail to express TNF-Rl suggesting that although TNFα loss may not be universal among TCL, protection from the TNFα-induced apoptosis may be quite common and involve diverse mechanisms. Because activated T lymphocytes are the prime target of TNFα (Abe et al., 2003, Blood 101 :1477-1483; van Horssen et al., 2006,

Oncologist. 11 : 397-408), it is believed that an escape from this control mechanism may be one of the critical properties T cells need to acquire in order to effectively undergo malignant cell transformation. The observation that subjects with various autoimmune disorders chronically treated with TNFα antagonists display an increased risk of developing lymphomas (Brown et al., 2002, Arthritis Rheum 46:3151-3158), particularly a hepatosplenic T-cell lymphoma in the setting of inflammatory bowel disease (Mackey et al., 2007, J Pediatr Gastroenterol Nutr 44:265-267; Zeidan et al., 2007, Leuk Lymphoma 48:1410-1413; Drini et al., 2008, Med J Aust 189:464-465), indirectly supports this notion.

The mechanisms underlying the TNFα gene promoter DNA methylation in ALK+ TCL are currently unclear. To determine if the NPM/ALK oncogene may be involved, the SUDHL-I cells were treated with an ALK specific siRNA for 72 hrs. However, no change in the status of the TNFα gene promoter methylation was seen. The fact that a subset of the ALK+ TCL cell lines does not show any methylation of the promoter (Figure 3A) also argues against a direct causal

PHIP/ 832722.1 38 relationship between the NPM/ ALK expression and the methylation of the TNFα promoter.

The results presented herein indicate that activation of the TNFα gene or administration of an exogeneous TNFα may have therapeutic beneficial effects in ALK+ TCL subjects. 5-ADC (available under the trade name of Dacogen™) and another structurally-related DNMT inhibitor 5'-aza-cytidine (Vidaza™) have already been successfully applied in subjects with myelodysplastic syndrome and chronic and acute myeloid leukemia, with the response rate reaching 60-70% for myelodysplastic syndrome (Claus et al., 2003, Oncogene 22: 6489-6496; Momparler, 2005, Semin Hematol 42: S9-S16; Momparler, 2005 Semin Oncol 32: 443-451). These cytidine analogs become incorporated into the DNA of dividing cells and trap DNMTs, especially DNMTl, by forming covalent bonds (Momparler, 2005, Semin Hematol 42: S9-S16). Other kinds of DNMTl inhibitors including the ones that block the enzymatic activity without the need of becoming incorporated into the DNA, are also being evaluated. Given that ALK+ TCL display the silencing of at least several tumor suppressor genes (Wasik et al., 2009, Semin Oncol 36 (2 Suppl 1):S27-S35;

Nagasawa et al., 2006, Leuk Res 30:303-312; Khoury et al., 2004, Blood 104:1580- 1581; Honorat et al., 2006, Blood 107:4130-4138; Zhang et al., 2005, Proc Natl Acad Sci USA 102:6948-6953; Zhang et al., 2007, Nature Med. 13: 1341-1348), there is a strong rationale to consider DNMT inhibition as a novel therapeutic option for this type of lymphoma.

Treatment of ALK+ TCL subjects with recombinant TNFα represents another possibility. Although the systemic administration of this cytokine has been found so far to be associated with high toxicity, regional therapy, primarily in the form of isolated limb perfusion, has proven very successful in treatment of nonresectable sarcomas and advanced melanomas (van Horssen et al., 2006,

Oncologist. 11 :397-408). In combination with melphalan, TNFα yields an overall response rate of over 75% for sarcomas and over 95% for melanomas providing a powerful argument in favor of TNFα as a highly effective therapeutic agent in cancer subjects. Interestingly, it has been suggested that TNFα efficacy in these

malignancies is in part due to increasing the permeability of the tumor-induced blood vessels and, consequently, selectively augmenting concentration of the

chemotherapeutic agent within the tumor tissue. Several different approaches have

PHIP/ 832722.1 39 recently been developed to improve the therapeutic index of systemically

administered TNFα, including conjugation with molecules against tumor- associated cell surface antigens and gene therapy with replication deficient viral vectors. This latter approach seems particularly promising and is currently evaluated in several different gastrointestinal cancers in stage II and III clinical trials (Zidi et al., 2009

Med Oncol Mar 11. [Epub ahead of print]). Future studies may include using engineered immune T lymphocytes specific for the tumor associated antigen as already successfully accomplished for the B-cell lymphoma marker CD20 (Till et al., 2008, Blood 112:2261-2271), co-transfected with the TNFα gene. Considering that ALK+ TCL strongly and quite selectively express the CD30 receptor, targeting the lymphoma cells by CD30 antibody-TNFα complexes or CD30-specific immune T lymphocytes expressing TNFα may prove very beneficial. Because TNFα is even more potent in its cell membrane-bound as compared to the soluble form (Abe et al., 2003, Blood 101 : 1477- 1483), this latter approach appears particularly attractive.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

PHIP/ 832722 1 40

Claims

1. A method for identifying a marker of response to cancer therapy in a mammal, the method comprises detecting hypermethylation of TNFα promoter compared to the methylation state of TNFα promoter from a biological sample derived from an otherwise normal mammal, present or increased expression of type 1

TNFα receptor (TNF-Rl) compared to the expression of TNF-Rl from a biological sample derived from an otherwise normal mammal, or a combination thereof; further wherein detection of said marker identifies said marker of response to cancer therapy.

2. The method of claim 1, wherein said cancer is lymphoma

3. The method of claim 1, wherein said biological sample is selected from the group consisting of a tumor tissue or a bodily fluid.

4. The method of claim 1, wherein said bodily fluid is peripheral blood.

5. The method of claim 1, wherein said therapy is TNFα therapy.

6. The method of claim 5, wherein said TNFα therapy comprises inducing expression of TNFα through reactivation of TNFα epigenetically silenced gene, inducing expression of exogenous TNFα, administration of exogenous TNFα cytokine, or any combination thereof.

7. A method of diagnosing cancer in a patient, said method comprising detecting a cancer marker in a biological sample derived from said patient, wherein said marker is selected from the group consisting of hypermethylation of the TNFα promoter compared to the methylation state of TNFα promoter from a biological sample derived from an otherwise normal patient, present or increased expression of type 1 TNFα receptor (TNF-Rl) compared to the expression of TNF-Rl from a biological sample derived from an otherwise normal patient, and any combination

PHIP/ 832722.1 41 thereof, further wherein detection of said cancer marker is an indication that said patient has cancer.

8. The method of claim 7, wherein said cancer is lymphoma.

9. The method of claim 7, wherein said biological sample is selected from the group consisting of a tumor tissue or a bodily fluid.

10. The method of claim 7, wherein said bodily fluid is peripheral blood.

11. A method of managing treatment of a cancer patient, said method comprising detecting a cancer marker from a biological sample derived from said patient, wherein said marker is selected from the group consisting of

hypermethylation of the TNFα promoter compared to the methylation state of TNFα promoter from a biological sample derived from an otherwise normal patient, present or increased expression of type 1 TNFα receptor (TNF-Rl) compared to the expression of TNF-Rl from a biological sample derived from an otherwise normal patient, and any combination thereof, further wherein detection of said cancer marker is an indication that said patient is capable of responding to TNFα therapy.

12. The method of claim 11, wherein said cancer is lymphoma.

13. The method of claim 11, wherein said biological sample is selected from the group consisting of a tumor tissue or a bodily fluid.

14. The method of claim 11 , wherein said bodily fluid is peripheral blood.

15. A method for identifying a cell that exhibits unregulated growth, said method comprising measuring the methylation status of a gene in a test cell and a control cell, wherein said control cell exhibits regulated growth; and comparing the methylation status of said gene from said test cell and said control cell, wherein a

PHIP/ 832722 1 42 hypermethylation status of said gene in said test cell compared to said control cell is an indication that said test cell exhibits unregulated growth.

16. A method for identifying a cell that is predisposed to unregulated growth, said method comprising measuring the methylation status of a gene in a test cell and a control cell, wherein said control cell exhibits regulated growth; and comparing the methylation status of said gene from said test cell and said control cell, wherein a hypermethylation status of said gene in said test cell compared to said control cell is an indication that said test cell is predisposed to unregulated growth.

PHIP/ 832722.1 43