BACKGROUND

The Insulinoma-Glucagonoma (IG20) gene plays an important role in cancer cell proliferation, apoptosis and survival (Chow & Lee (1996)DNA Seq.6:263-273; Chow, et al. (1998)Genome41:543-552; Schievella, et al. (1997)J. Biol. Chem.272:12069-12075; Brinkman, et al. (1999)J. Biol. Chem.274:30882-30886; Murakami-Mori, et al. (1999)J. Immunol.162:3672-3679; Telliez, et al. (2000)Biochem. Biophys. Acta1478:280-288; Al-Zoubi, et al. (2001)J. Biol. Chem.276:47202-47211; Lim & Chow (2002)Mol. Carcinog.35:110-126; Efimova, et al. (2004)Oncogene23:1076-1087; Efimova, et al. (2003).Cancer Res.63:8768-8776; Lim, et al. (2004)Int. J. Cancer109:24-37; Ramaswamy, et al. (2004)Oncogene23:6083-6094). Additionally, it plays an important role in neurotransmission (Zhang, et al. (1998)Proc. Natl. Acad. Sci. USA95:2586-2591; Tanaka, et al. (2001)Mol. Biol. Cell12:1421-1430; Yamaguchi, et al. (2002).Proc. Natl. Acad. Sci. USA99:14536-14541), neurodegeneration (Del Villar & Miller (2004)Proc. Natl. Acad. Sci. USA101:4210-4215) and guanine nucleotide exchange (Wada, et al. (1997)J. Biol. Chem.272:3875-3878; Brown & Howe (1998)Curr. Biol.8:R191; Iwasaki & Toyonaga (2000)EMBO J.19:4806-4816; Levivier, et al. (2001)Biochem. Biophys. Res. Commun.287:688-695). These divergent functions are most likely mediated through alternative splicing of the IG20 gene to provide splice variants and protein diversity (Chow, et al. (1998)Genome41:543-552; Al-Zoubi, et al. (2001)J. Biol. Chem.276:47202-47211; Efimova, et al. (2004)Oncogene23:1076-1087). Down-regulation of all endogenous IG20 splice variants, using anti-sense oligonucleotides, has been shown to result in spontaneous apoptosis of cancer cells in vitro and in vivo, but not in normal cells (Lim & Chow (2002)Mol. Carcinog.35:110-126; Lim, et al. (2004)Int. J. Cancer109:24-37).

U.S. Pat. No. 8,722,637 describes “IG20 and IG20-[SV2], and the previously reported KIAA0358, MADD, and DENN-SV are splice variants of the IG20 gene, which is localized to chromosome 11p11 and consists of 36 exons. Differences among the above variants are due to alternative splicing of exons 13L, 16, 21, 26 and 34.” The IG20, MADD, SV2 and DENN-SV isoforms may be considered to be described by Adeeb, et al. (2001)J. Biol. Chem.276(50):47202-47211). Subsequent to the disclosure in Al-Zoubi, et al., longer isoforms were identified, including KIAA0358. Consequently, the IG20 splice variant identified by Al-Zoubi, et al. as IG20 became known as IG20pa because, as U.S. Pat. No. 8,722,637 explains, the IG20 expression protein is a pro-apoptotic signaling molecule which is distinct from the IG20-FL or full-length variant (col. 5, Ins. 43-45).

It has further been suggested that all seven variants of IG20 identified to date arise from alternative splicing of exons 13L, 16, 21, 26 and 34. The full-length cDNA of IG20 (IG20-FL) (accession number AF440100) is 5995 base pairs (bps) long, consists of all 36 exons and represents the longest variant (Efimova, et al. (2004)Oncogene23:1076-1087). These splice variants may be graphically represented as inFIG. 1. Splicing of exon 34 alone generates KIAA0358 (accession number AB002356) that consists of 5942 bps. Splicing of exons 21 and 26, and splicing of exons 16, 21 and 26 generate 5878 bps long IG20 accession number AF440101) and 6002 bps long MADD (accession number U77352), respectively. MADD is also known as DENN (accession number U44953) that is 5844 bps long. Splicing of exons 13L, 21 and 26, and 13L, 16, 21 and 26 generate 5749 bps long IG20-SV2 (accession number AF440102) and 5689 bps long IG20-SV3 (accession number AF440103) (earlier referred to as DENN-SV). Finally, splicing of all five exons (13L, 16, 21, 26 and 34) generates IG20-SV4 (accession number AF440434), which is the shortest variant and consists of 5619 bps.

At the time of filing of U.S. Pat. No. 8,722,637, it was determined that IG20pa, MADD, IG20-SV2 and DENN-SV are expressed in human tissues and, to a greater degree, in tumors (U.S. Pat. No. 8,722,637 at col. 5, lns. 1-2). Overexpression of DENN-SV was associated with enhanced cell replication and resistance to apoptosis induced by chemotherapy (U.S. Pat. No. 8,722,637 at col. 5, lns. 4-6). Overexpression of IG20pa was shown to override endogenous DENN-SV function, resulting in apoptosis (U.S. Pat. No. 8,722,637 at col. 5, lns. 16-17).

Through the experimentation performed in U.S. Pat. No. 8,722,637, it was determined that, “[b]ecause DENN-SV lacks both exons 13L and 16, MADD lacks exon 16 and IG20-SV2 lacks exon 13L, these results demonstrated that expression of both exons 13L and 16, as seen in IG20[pa], is required for anti-proliferative and pro-apoptotic properties, whereas deletion of both exons, as seen in DENN-SV, is required for pro-proliferative and anti-apoptotic properties.” U.S. Pat. No. 8,722,637 at col. 17, lns. 38-44. Ultimately, U.S. Pat. No. 8,722,637 concludes that “[t]here is clear evidence to suggest that IG20[pa] and DENN-SV have contrasting effects on apoptosis and cell proliferation.” U.S. Pat. No. 8,722,637 at col. 18, lns. 37-39. It is further concluded that “IG20 [pa] is a pro-apoptotic protein that can interact with DR4 and DR5 and significantly enhance TRAIL induced apoptosis by facilitating DISC formation with increased recruitment of FADD and caspase-8.” U.S. Pat. No. 8,722,637 at col. 23, lns. 1-4. Finally, it was concluded that “IG20[pa] can render cells more susceptible to apoptosis and suppress cell growth. This raises the possibility of using IG20[pa] to render cells that are otherwise resistant to become more susceptible to various modalities of cancer therapy.” U.S. Pat. No. 8,722,637 at col. 27, lns. 20-24.

As a result of the understanding achieved through over-expression of IG20 splice variants, it was concluded that “[c]ells transfected with IG20[pa] and DENN-SV were most susceptible and resistant to TNFα-induced apoptosis respectively, whereas cells transfected with MADD or IG20-SV2 did not show significant differences relative to cells transfected with a control plasmid. Because DENN-SV lacks both exons 13L and 16, MADD lacks exon 16 and IG20-SV2 lacks exon 13L, these results demonstrated that expression of both exons 13L and 16, as seen in IG20[pa], is required for anti-proliferative and pro-apoptotic properties, whereas deletion of both exons, as seen in DENN-SV, is required for pro-proliferative and anti-apoptotic properties.” U.S. Pat. No. 8,722,637 at col. 17, lns. 34-44.

Therefore, prior to U.S. Pat. No. 8,722,637, the full complexity of IG20 splice variant expression, selective expression of different isoforms in different tissues and the unique functional attributes of each of the splice variants were not appreciated. Based on the experimentation into the over-expression of IG20 splice variants as disclosed in U.S. Pat. No. 8,722,637, the impact of indiscriminate knock-down of splice variants, not taking into account variant splicing at introns 16 (MADD), 13L and 16 (DENN-SV), 21 and 26 (MADD, DENN-SV, IG20pa) and 34 (KIAA0358, IG20-SV4), could prove vital for normal neuronal function and survival of the animal (i.e., KIAA0358), cancer cell survival (i.e., MADD) and proliferation (i.e., DENN-SV). Therefore, the patent provides a functional characterization of IG20 isoforms and explains the complexity in devising strategies which may selectively modulate various isoform expression while avoiding unintended lethal consequences.

The IG20pa splice variant is pro-apoptotic, anti-proliferative, and renders cells more susceptible to induced cell death (i.e., is a tumor suppressor). IG20pa, or a fragment thereof, may be overexpressed to control cell proliferation, cell cycle, and to render cells more susceptible to chemotherapy, radiation therapy or death receptor mediated cell death.

DENN-SV expression can be down-modulated to reduce cell proliferation, affect cell cycle and increase susceptibility to treatment with chemotherapy, radiation therapy and death receptor mediated cell death.

Differential expression of IG20pa and DENN-SV splice variants renders cells either more susceptible or resistant to induced cell death respectively, and the pro-apoptotic property of IG20pa variant may be exploited to render tumor cells that are otherwise chemotherapeutic resistant to become susceptible to killing by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and/or chemotherapeutic agents.

When IG20pa was over-expressed in cells, the cells showed significantly reduced proliferation and were much more susceptible to spontaneous TNFα- and TRAIL-induced apoptosis. Thus, the pro-apoptotic property of the IG20pa splice variant may be exploited to render tumor cells which are otherwise resistant to become susceptible to killing by TRAIL and/or chemotherapeutic agents.

Down-modulation of expression of IG20 splice variants with siRNA was evaluated using siRNA targeting the mid-region of the IG20 mRNA, specifically exon 15, having the sequence (5′-GTACCAGCTTCAGTCTTTC-3′; SEQ ID NO:14) and siRNA targeting the Death Domain (DD) region of IG20 mRNA. Both the mid-region and the DD region are present in all IG20 splice variants.

Treatment of cells with siRNA against the mid-region of IG20 mRNA, but not against the death domain (DD) of IG20 mRNA suppresses the levels of DENN-SV (completely abrogates). Cells treated with siRNA against the mid-region, i.e., exon 15, of IG20 mRNA undergo spontaneous apoptosis. Moreover, the cells that fail to undergo spontaneous apoptosis after siRNA treatment against the mid-region, i.e., exon 15, of IG20 mRNA are more susceptible to TNF-α induced apoptosis.

It may be concluded that, to decrease cell replication, selection of siRNA to down-modulate DENN-SV expression, specifically, without affecting expression of IG20 splice variants which are necessary for normal cell function, in particular, neuronal function and survival (KIAA0358 and IG20-SV4 splice variants), is desired.

Moreover, contrasting prior art oligodeoxynucleotide sequences, which in many cases proved lethal, oligodeoxynucleotides which take into account the research of U.S. Pat. No. 8,722,637 may be optimized to bind to particular exons which are differentially expressed in different isoforms. Knockdown using such engineered oligodeoxynucleotides may knockdown only those isoforms in which that particular targeted exon is expressed, allowing for selective knockdown of intended isoforms and reducing unintended negative effects associated with knockdown of critical isoforms.

Map kinase Activating Death Domain (MADD) containing protein, a product of the MADD splice variant of the IG20 gene, is essential for cancer cell survival. MADD is expressed at much higher levels in cancer cells and tissues relative to their normal counterparts. MADD has been shown to bind to death receptor-4 (DR4) and death receptor-5 (DR5) and to confer resistance to TRAIL-induced apoptosis in thyroid, ovarian and cervical cancer cell lines (Mulherkar, et al. (2007)J. Biol. Chem.282:11715-11721; Subramanian, et al. (2009)J. Clin. Endocrinol. Metab.94:1467-1471; Prabhakar, et al. (2008)Clin. Cancer. Res.14:347-351; Li, et al. (2011)Am. J. Obstet. Gynecol.205:362 e312-325).

Abrogation of MADD, but not the other IG20 splice variants, may render cancer cells more susceptible to spontaneous as well as TRAIL-induced apoptosis. Spontaneous as well as TRAIL-induced apoptosis in cells devoid of MADD may be inhibited by expression of CrmA or dominant-negative FADD, thereby suggesting that endogenous MADD may interfere with caspase-8 activation. Further, it has been found that MADD can directly interact with death receptors, but not with either caspase-8 or FADD, but nonetheless inhibits caspase-8 activation. MADD has been shown to interfere with recruitment of FADD to the cytoplasmic domain of death receptors (Mulherkar, et al. (2007)J. Biol. Chem.282(16):11715-11721). This demonstrates the importance of MADD in the control of cancer cell survival/death and in conferring resistance to TRAIL-induced apoptosis.

The ERK (extracellular signal-related kinase) pathway is a drug target for cancer chemotherapy since, in approximately one-third of all human cancers, there is deregulation of the mammalian mitogen-activated protein kinase (MAPK) pathways leading to ERK activation. MAPKs are serine/threonine-specific protein kinases which respond to extracellular stimuli (mitogens) and regulate several important and critical cellular functions required for cell homeostasis like metabolism, cell cycle progression, expression of cytokines, motility and adherence. Hence MAPKs influence cell survival, proliferation, differentiation, development and apoptosis. Extracellular stimuli such as cytokines, growth factors and environmental stresses lead to the sequential activation of a signaling cascade composed of MAPKs.

When activated, ERK1/2 phosphorylates several nuclear and cytoplasmic substrates involved in a multitude of cellular processes, including transcriptional factors, signaling proteins, kinases and phosphatases, cytoskeletal proteins, apoptotic proteins and proteinases. Even though the ERK pathway may be activated by numerous extracellular signals, the pathways whereby cytokines and growth factors activate ERK signaling are of particular relevance to cancer. For example, TNF-α, a cytokine rich in tumor stroma binds to TNF receptor 1 (TNFR1) which is present on cancer cells and potently activates ERK MAPKs. In the absence of MADD this pro-survival signaling pathway may be converted into an apoptotic signaling pathway leading to cancer cell death (Kurada, et al. (2009)J. Biol. Chem.284:13533-13541).

An extrinsic cell death inducing signaling pathway may be initiated upon death ligand (e.g., TRAIL) binding to its cognate death receptors. The death receptors undergo trimerization and recruit FADD resulting in subsequent caspase-8 activation followed by executioner caspase-3 activation leading to apoptosis. TRAIL normally binds to DR4 and DR5 on cancer cells resulting in death receptor (DR) oligomerization and subsequent recruitment of FADD and procaspase-8 to the DRs (Bodmer, et al. (2000)Nat. Cell Biol.2:241-243; Sprick, et al. (2000)Immunity12:599-609; Kischkel, et al. (2000)Immunity12:611-620). Procaspase-8 then undergoes proximity induced cleavage and activation forming caspase-8 which then activates executioner caspase-3 which causes apoptotic cell death. However, in cancer cells where MADD is over-expressed, MADD binds to DR4 and DR5 and prevents FADD recruitment to the DRs. Upon MADD down-regulation, FADD is more readily recruited to the DRs, resulting in enhanced apoptosis (Mulherkar, et al. (2007)J. Biol. Chem.282:11715-11721; Mulherkar, et al. (2006)Oncogene25:6252-6261).

TRAIL is unique in that it generally does not adversely affect normal cells or tissues (Keane, et al. (1999)Cancer Res.59:734-741). However, development of chemotherapy and TRAIL resistance due to the interference of different anti-apoptotic proteins remains a major challenge. MADD is one such anti-apoptotic protein demonstrating the utility of MADD down-regulation in rendering cancer cells susceptible to cell death (Mulherkar, et al. (2006)Oncogene25:6252-6261).

MADD may have a dual function in regulating apoptosis depending on its phosphorylation by Akt. The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid phosphatase that negatively regulates the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. MADD can act as a pro-apoptotic factor to initiate apoptosis when its phosphorylation is attenuated by PTEN (Jayarama, et al. (2014)J. Cell. Biochem.115(2):261-270). TRAIL induces an up-regulation of PTEN with a concomitant reduction in MADD phosphorylation. Down-regulation of PTEN interferes with TRAIL-induced reduction in pMADD levels. Non-phosphorylated MADD translocates from the plasma membrane to cytoplasm where it binds to 14-3-3 protein and displaces 14-3-3 associated Bax, which Bax translocates to mitochondria resulting in cytochrome-c release. Taken together, one may conclude that PTEN can convey the death signal by preventing MADD phosphorylation by Akt.

The extrinsic apoptotic pathway may be abrogated by phosphorylated MADD. Endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing FADD recruitment. However, in cells susceptible to TRAIL treatment, TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and FADD association with DR4, which allows death-inducing signaling complex (DISC) formation leading to apoptosis (Li, et al. (2010)J. Biol. Chem.285(29):22713-22722). Thus, the pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co-targeting the Akt-MADD axis is likely to increase efficacy of therapeutics which involve DR4/5 binding, including TRAIL-based therapies.

The intrinsic apoptotic pathway is initiated when a death signal induces the release of mitochondrial pro-apoptotic proteins such as cytochrome-c (Li, et al. (1997)Cell91(4):479-489), mitochondrial apoptosis-inducing factor (Susin, et al. (1999)Nature397(6718):441-446) and Smac/Diablo (Du, et al. (2000)Cell102(1):33-42; Verhagen, et al. (2000)Cell102(1):43-53). Cytochrome-c forms a complex with Apaf-1 and procaspase-9 resulting in the activation of caspase-9. Smac/Diablo can associate with Inhibitor of Apoptosis Proteins (IAPs) and counteract their caspase inhibitory effects. The intrinsic pathway is regulated by the Bcl-2 family members. For example, in response to pro-apoptotic stimuli, the cytosolic Bax and Bad translocate to mitochondria to permeabilize the outer mitochondrial membrane leading to cytochrome-c release into the cytosol. In contrast, Bcl-2 and Bcl-xL can associate with Bax and Bad thereby preventing them from inducing death (Antignani & Youle (2006)Curr. Opin. Cell Biol.18(6):685-689). Interestingly, non-phosphorylated MADD translocates from the plasma membrane to cytoplasm where it binds to 14-3-3 and displaces 14-3-3 associated Bax, which translocates to mitochondria resulting in cytochrome-c release (Jayarama, et al. (2014)J. Cell. Biochem.115(2):261-270).

The MADD cDNA sequence is available on the GenBank database under accession number NM_130470, and is represented herein by the nucleotide sequence of SEQ ID NO:11 and the polypeptide sequence of SEQ ID NO:12. Interfering RNAs which down-regulate MADD, including siRNA, shRNA and antisense oligonucleotides, are designed to target a nucleic acid sequence of exon 13L of a splice variant of the IG20 gene, and include any allelic variants and naturally occurring mutants of MADD, and polymorphisms which occur in the MADD splice variant which may be found in a particular segment of the population. In other words, sequences which are highly similar (e.g., about 95% at the amino acid level and about 75% at the nucleic acid level) and which represent naturally occurring variations in the MADD splice variant are within the scope of the disclosure, wherein the siRNA, shRNA and antisense oligonucleotides disclosed herein are capable of down-regulating the expression of such sequences. Exon 13L of the MADD splice variant may comprise a nucleotide sequence represented by nucleotides 2699 to 2827 of SEQ ID NO:11. Nucleic acid sequences which are about 80% or 90% or 95% similar at the nucleic acid level to the MADD sequence disclosed herein may also be down-regulated. Nucleic acid sequences which generate siRNA and shRNA which comprise nucleic acid sequences complementary to a nucleic acid sequence of exon 13L of the MADD splice variant of the IG20 gene and/or an mRNA transcript of exon 13L of the MADD splice variant, as well as nucleic acid variations which may occur within the exon 13L target region are within the scope of the instant disclosure. Moreover, antisense oligonucleotides which comprise nucleic acid sequences complementary to a nucleic acid sequence of exon 13L of the MADD splice variant of the IG20 gene and/or an mRNA transcript of exon 13L of the MADD splice variant, as well as nucleic acid variations which may occur within the exon 13L target region are within the scope of the instant disclosure

Methods for specifically down-regulating the expression of a splice variant of an IG20 gene have been shown to include: (a) obtaining a nucleic acid molecule which is capable of down-regulating MADD expression, wherein the nucleic acid molecule or a transcription product thereof is capable of selectively binding to an mRNA molecule, the mRNA molecule which includes a nucleic acid sequence of a MADD splice variant of the IG20 gene; and (b) contacting a cell which expresses the MADD splice variant of the IG20 gene with the nucleic acid molecule, wherein the nucleic acid molecule down-regulates the expression of the MADD splice variant. In the cytoplasm, nucleic acids selected from siRNA, expressed shRNAs, and antisense oligonucleotides bind to target exon 13L mRNA and lead to degradation of the target 13L mRNA which down-regulates expression of the MADD splice variant.

Specifically, down-regulating MADD expression has been shown to be a substantial downregulation, for example, more than 90% or 95% reduction of the endogenous MADD expression. In fact, downregulation of, for example, at least 40%, at least 50%, at least 60%, at least 70%, and at least 80% of endogenous MADD expression is desirable.

Isolated siRNA, shRNA and antisense oligonucleotides which selectively down-regulate the expression of a splice variant of an IG20 gene, wherein the splice variant is MADD, are disclosed in U.S. Pat. No. 7,910,723. The patent describes the development of “splice variant specific” nucleic acid molecules that selectively down-regulate one or more of the splice variants of the IG20 gene and their relative importance in cancer cell survival. Not all cancer cells express all IG20 splice variants and some express only the MADD and the DENN splice variants. Using cells which expressed four known splice variants or only the MADD and DENN splice variants, and different nucleic acid molecules selected from shRNAs, siRNAs and antisense oligonucleotides, which specifically targeted exon 13L (to down-regulate IG20pa and MADD), exon 16 (to down-regulate IG20pa and IG20-SV2) and exon 15 (exon 15 is expressed in all splice variants of the IG20 gene and therefore it can down-regulate expression of all splice variants), the critical requirement for MADD for cancer cell survival has been demonstrated. Although siRNA, shRNA and antisense oligonucleotides targeting exon 16 could knockdown IG20pa, only the siRNA, shRNA and antisense oligonucleotides targeting exon 13L could cause cancer cell death. This indicated for the first time that specifically MADD might be critical for cancer cell survival. An additional line of evidence which shows MADD to be essential and sufficient for cancer cell survival is demonstrated in cells transfected with Mid-shRNA resistant IG20 splice variants in which the 3rdbase of the triple codons was replaced in the Mid-shRNA targeted region of the cDNA constructs, which DNA substitutions will not alter the amino acid sequence but renders them resistant to Mid-shRNA). When the expression of all endogenous IG20 splice variants was down-regulated using Mid-shRNA, only cells expressing a MADD splice variant could prevent cell death, whereas the other splice variants could not prevent cell death. Using cancer cells that expressed only MADD and DENN-SV splice variants, it was shown that MADD could be selectively down-regulated using siRNA, shRNA or antisense oligonucleotides which target exon 13L and it was shown that MADD, alone, is sufficient and required for cancer cell survival (Mulherkar, et al. (2006)Oncogene25:6252-61).

Thus, none of the earlier research revealed the full complexity of IG20 gene expression, the differential expression of the splice variants in different tissues, the unique functional attributes of each of the splice variants (i.e., cancer cell survival (MADD) and proliferation (DENN). Therefore, it is the discovery of various splice variants, their functional characterization and the ability to identify specific nucleic acid molecules which can selectively down-regulate expression of splice variants which allowed the inventors to develop cancer therapeutics without unintended potential negative consequences.

U.S. Pat. No. 7,910,723 describes nucleic acid molecules which target exon 13L of the IG20 gene and the use of encoded siRNA, shRNA and antisense oligonucleotides in down-regulating expression of MADD protein. The patent describes how the nucleic acid selection may be optimized to achieve effective down-regulation of MADD expression, including selecting for nucleic acid molecules which consist essentially of a nucleotide sequence CGGCGAATCTATGACAATC (SEQ ID NO:1), and transcribed products thereof, encoding nucleic acid molecules consisting essentially of a nucleotide sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO:2). Species nucleic acid molecules representative of such strategy include siRNA, shRNA and antisense oligonucleotides, which comprise less than 50% GC nucleotide content, high AU nucleotide content toward the 3′ end and no inverted repeats within the siRNA region, which oligonucleotides constitute an array of nucleic acid molecules which may span the exon 13L nucleotide sequence. These encoded nucleic acid molecules and encoded siRNA, shRNA and antisense oligonucleotides are demonstrated to be sufficient to down-regulate the expression of MADD splice variants. Natural variations of MADD including specific SNPs, allelic variants, or mutations which may appear in one or more of sub-groups of cancer types may be targeted by such nucleic acid molecules.

Methods for down-regulating expression of MADD are described to include: (a) obtaining a nucleic acid molecule which selectively down-regulates MADD expression, wherein the nucleic acid molecule is capable of selectively binding to an mRNA molecule of a MADD splice variant of the IG20 gene; and (b) contacting a cancer cell which expresses the MADD splice variant of the IG20 gene with the nucleic acid molecule, wherein the nucleic acid molecule down-regulates the expression of the MADD splice variant in the cancer cell.

Moreover, representative nucleic acid molecules which are shown to down-regulate the MADD splice variant of IG20, allelic variations thereof, polymorphisms thereof, and genetic mutations thereof, include siRNA, shRNA, and anti-sense nucleic acid molecules which may comprise a nucleotide sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO:2). The siRNA may be in the form of a duplex with the cognate antisense nucleic acid, which cognate antisense nucleic acid is complementary to a target MADD exon 13L nucleotide sequence and/or mRNA transcripts of MADD exon 13L.

Down-regulation of specific splice variants of the IG20 gene is difficult because of the very short target sequences which are differentially expressed in different splice variants. U.S. Pat. No. 7,910,723 explains that, in an embodiment of the invention, down-regulation may be achieved through the use of specially designed short hairpin RNA molecules (shRNA). shRNA comprise complementary sense and antisense sequences of the target gene linked by a loop structure. A dsDNA may be cloned into an expression vector for transfection which may then be transcribed to form shRNA, which is cleaved into siRNA, which may inhibit gene expression through RNA interference (RNAi). In an embodiment, nucleic acids encoding such an shRNA, including the structure:
Xsense−hairpin loop−Xanti-sense,
wherein X (encoding siRNA) includes or consists essentially of a nucleic acid having the sequence CGGCGAATCTATGACAATC (SEQ ID NO:1). The nucleic acid is transcribed to form shRNA and may be cleaved to form siRNA which may ultimately inhibit MADD expression. Thus, RNA molecules which are transcribed in vitro or in vivo, e.g., in a cancer cell or tumors to form shRNA and siRNA are also included.

An exemplary dsDNA nucleic acid sequence encoding an shRNA inhibiting MADD expression may be CGGCGAATCTATGACAATCTTCAAGAGAGATTGTCATAGATTCGCCG (SEQ ID NO:13), wherein the hairpin loop region between Xsenseand Xanti-senseis from positions 20-28 of the sequence. The hairpin loop region may contain any suitable sequence. To construct shRNA vectors, see McIntyre & Fanning (2006)BMC Biotechnol.6:1.

In an embodiment, double-stranded RNA or dsRNA refers to a double-stranded RNA which matches a predetermined gene sequence which is capable of activating cellular enzymes which degrade corresponding messenger RNA transcripts of the gene. These dsRNAs comprise nucleic acid molecules which may be short interfering RNA (siRNA) and may be used to inhibit gene expression. The term “double-stranded RNA” or “dsRNA,” as used herein, refers to a double-stranded RNA molecule capable of RNA interference “RNAi,” including small interfering RNAs “siRNA.”

siRNA, shRNA and antisense oligonucleotide molecules as described herein may also include nucleic acid modifications known in the art to enhance stability and to enhance cleavage destruction of the target mRNA. With respect to the embodied nucleic acid molecules, a representative example may comprise a 19-base core which includes 2 or 3 nucleotide overhanging 3′ ends, such as a 3′ terminal thymidines (TT). The overhangs may play a structural role for presenting a symmetrical duplex to the RNA-Induced Silencing Complex (RISC). Often dTdT is selected because it can confer nuclease resistance to nucleic acid molecules. Nevertheless, some investigators prefer UU overhangs or overhangs that are complementary to the authentic mRNA target. What is most important is the identity of the 19-base core of the nucleic acid molecules towards a unique mRNA target.

The siRNA, shRNA and antisense oligonucleotide molecules may also be chemically synthesized de novo. The synthesized nucleic acid molecules may be in the form of a single-stranded nucleic acid molecule or may be in the form of a duplex with the cognate antisense nucleic acid molecule. The siRNA, shRNA and antisense oligonucleotides comprise a nucleic acid sequence which is complementary to a target MADD exon 13L nucleotide sequence and/or mRNA transcripts of MADD exon 13L.

RNA interference is a conserved pathway found in most eukaryotes where dsRNAs down-regulate expression of genes with complementary sequences. Long dsRNAs are degraded by the endoribonuclease Dicer into small effector molecules called siRNAs. siRNAs are usually around 21 base pairs (bp) long with a central 19 bp duplex and 2-base 3′-overhangs (this could be TT) (Elbashir, et al. (2001)Genes&Dev.15:188-200). In mammals, Dicer processing occurs in a multiprotein complex with the RNA-binding protein TRBP. The nascent siRNA associates with Dicer, TRBP, and Argonaute 2 (Ago2) to form the RISC. Once in RISC, one strand of the siRNA (the passenger strand/the strand that has the same sequence as the target mRNA) is degraded or discarded while the other strand (the guide strand/strand that is complementary to the targeted mRNA) remains to direct sequence specificity of the silencing complex. The Ago2 component of RISC is a ribonuclease that will cleave the target RNA under direction of the guide strand. Once the RISC complex is activated, it can move on to target additional mRNA targets. This effect amplifies gene silencing and allows the therapeutic effect to last for 3-7 days in rapidly dividing cells. Many researchers today employ synthetic RNA duplexes as their RNAi reagents, which mimic the natural siRNAs that result from Dicer processing of long substrate RNAs. These synthetic siRNA duplexes are transfected into cell lines where they mimic in vivo Dicer products.

The modulation of MADD expression and/or downregulation of MADD expression utilizing siRNA, shRNA or antisense oligonucleotides may augment traditional cancer therapies.

From a chemotherapeutic perspective, cancer chemotherapy has advanced dramatically in recent years. Numerous cancer chemotherapy substances have been identified which are effective in treating cancer. Nonetheless, many cancer chemotherapies are characterized by toxic side effects which are often encountered with administration of particular chemotherapeutics.

For example, the administration of many established chemotherapeutics is well-known to result in unwanted side-effects including nausea and vomiting, loss of appetite, change in taste, thinned or brittle hair, joint pain, nail change, and tingling in the hands or toes. More serious side effects such as impaired bowel movement, difficulty in swallowing, dizziness, shortness of breath, severe exhaustion, and chest pain may also occur. Consequently, there is an unmet need to provide care givers with a treatment regimen which is possessed of the advantages of the chemotherapeutic potential of various known chemotherapeutics, but without the unwanted side effects which limit compliance and efficacy.

Chemotherapeutics, such as protein kinase inhibitors, have been administered in the treatment of various forms of chronic myeloid leukemia, breast cancer, lung cancer, colorectal cancer, primary kidney cancer, liver cancer and thyroid cancer, and other types of solid tumor cancers as well as Kaposi's sarcoma with a good degree of success. Protein kinase inhibitors may be characterized as types of enzyme inhibitors which block the action of protein kinases which act by adding a phosphate group to a protein, thereby modulating its function. The protein kinases add phosphate groups to serine, threonine, or tyrosine amino acids on the protein. Most kinases act on both serine and threonine, tyrosine kinases act on tyrosine, and dual-specificity kinases act on all three. There are a few protein kinases that can phosphorylate other amino acids such as histidine kinases phosphorylate histidine residues. Protein kinase inhibitors act by directly interacting with the ATP binding site or by altering the kinase conformation to prevent productive ATP binding. Protein Kinase Inhibitors can be allosteric inhibitors, protein substrate competitive inhibitors, ATP competitive inhibitors or covalent bond forming inhibitors.

Representative protein kinase inhibitors include Imatinib, trastuzumab, bevacizumab, gefitinib, cetuximab, Sorafenib. Imatinib is an inhibitor of c-Abl and is used in the treatment of chronic myeloid leukemia. Trastuzumab is an inhibitor of HER2 and is used in the treatment of breast cancer. Bevacizumab is an inhibitor of vascular endothelial growth factor receptor and is used in the treatment of metastatic colorectal cancer. Gefitinib and Cetuximab are inhibitors of EGF receptors and used in the treatment of lung and colorectal cancer. Sorafenib is a small inhibitor of various tyrosine protein kinases such as VEGFR, PDGFR, and Raf family kinases, and used in the treatment of advanced renal cell carcinoma, hepatocellular carcinoma and radioactive iodine resistant advanced thyroid cancer. Erlotinib, like gefitinib, inhibits EGFR. Lapatinib is a dual inhibitor of EGFR and a subclass called Human EGFR type 2. EGFR is not the only growth factor targeted. Sunitinib is multi-targeted, inhibiting PDGFR and VEGF. Nilotinib inhibits the fusion protein bcr-abl and is typically prescribed when a patient has shown resistance to imatinib. More protein kinase inhibitors are currently in development. Three TKIs are currently showing promise in clinical trials. Bosutinib targets abl and src kinases. Neratinib, like lapatinib, inhibits EGFR and Human EGFR type 2. Vatalanib inhibits both VEGFR and PDGFR.

Protein kinase inhibitors also pose adverse effects on patients such as cardiovascular and dermatological toxicities. Myelosuppression and neutropenia are the most common adverse effects associated with c-Abl inhibitors such as Imatinib. For the EGFR-targeted inhibitors, most commonly observed side effects are fatigue, diarrhea, and the development of various dermatological toxicities such as acne-form-like rash, and hand-foot syndrome.

Chemotherapeutics, such as anthracyclines, are used in the treatment of various solid tumors and hematological malignancies. Anthracyclines work by inhibiting DNA and RNA synthesis by intercalating between base pairs of the DNA/RNA strand and thereby, preventing the replication of rapidly dividing cancer cells. They also work by inhibiting topoisomerase II enzyme, thereby preventing the relaxing of supercoiled DNA and hence, blocking DNA transcription and replication. Anthracyclines are among the most commonly used chemotherapeutic agents. The effect of anthracyclines in treating cancer could be attributed to their cytostatic and cytotoxic actions such as free radical formation, lipid peroxidation and direct membrane effects. The best characterized mechanisms are the interactions with the DNA topoisomerase II or DNA itself via intercalation or covalent binding formation and modification of the bases, which are responsible for disturbances in DNA replication and transcription, eventually leading to induction of apoptotic cell death. Anthracyclines are used in the treatment of cancers including leukemias, lymphomas, breast, stomach, uterine, ovarian, bladder cancer and lung cancers.

Representatives of anthracyclines include daunorubicin, doxorubicin, epirubicin, idarubicin and valrubicin. Daunomycin (daunorubicin) was the first anthracycline compound to be characterized structurally and stereochemically. Daunorubicin is used in treating acute lymphoblastic and myeloblastic leukaemias. Adriamycin (generic name doxorubicin) is a hydroxyl derivative of daunorubicin. Doxorubicin is one of the most widely used chemotherapeutic agents and is generally prescribed in combination with other drugs. Doxorubicin has a broad spectrum of activity. It is one of the most effective drugs for solid tumor treatment, e.g., breast cancer, small cell lung cancer and ovarian carcinoma treatments. It has significant activity against bladder, stomach, liver and thyroid tumors, Ewing's and osteogenic bone tumors, soft tissue sarcoma, neuroblastoma and Wilms tumor. It is also active against multiple myeloma, several types of leukemia and cutaneous T-cell lymphoma. It also plays an important role in treatment of Hodgkin's disease and non-Hodgkin's lymphomas. Epirubicin is an epimer of doxorubicin and differs only in the orientation of the C-4 hydroxyl group on the sugar. Because of this slight change in the structure, epirubicin has lower cardiotoxicity than doxorubicin. Epirubicin is used in the treatment of gastric and breast cancer and is also indicated for the treatment of carcinoid, endometrial, lung, ovarian, esophageal and prostate cancers as well as soft tissue sarcomas. Idarubicin is an analog of daunorubicin. It lacks the C-4 methoxy group and this increases its lipophilicity. Idarubicin has improved activity as induction therapy for acute myelogenous leukaemia. Valrubicin is N-trifluoroacetyl, 1-4-valerate derivative of doxorubicin. Valrubicin enters cells more rapidly than doxorubicin. It is used specifically in the treatment of early bladder cancer.

One of the major side effects of anthracyclines is the cardiotoxicity, which often presents as ECG changes and arrhythmias, or as a cardiomyopathy leading to heart failure. It is related to a patient's cumulative lifetime dose and therefore, treatment is usually stopped upon reaching the maximum cumulative dose of that particular anthracycline. Cardiotoxicity is further overcome by using Dexrazoxane, a cardioprotectant, or by using liposomal formulations of daunorubicin and doxorubicin or by using longer infusion rates.

Chemotherapeutics, such as nucleoside analogs, are synthetic, chemically-modified compounds that mimic their physiological counterparts and thereby interfere with the synthesis of nucleic acids by incorporating into DNA and RNA. They can also interact and inhibit essential enzymes such as DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, RNA-dependent RNA polymerase, kinases, DNA methyltransferases, pyrimidine and purine nucleoside phosphorylase etc. Nucleoside analogues exert cytotoxic effect by being incorporated into and altering the DNA and RNA macromolecules themselves, by interfering with various enzymes involved in synthesis of nucleic acids, or by modifying the metabolism of physiological nucleosides.

Representative nucleoside analogues, such as Gemcitabine, 5-Fluorouracil and Cytarabine, are used in the treatment of breast cancer, non-small cell lung cancer, pancreatic cancer, bladder cancer, biliary tract cancer and colon cancer, esophageal cancer, stomach cancer, pancreatic cancer, breast cancer, cervical cancer, and other types of solid tumor cancers. The triphosphate analogue of gemcitabine replaces cytidine during DNA replication leading to arrest of tumor growth. 5-Fluorouracil acts as a thymidylate synthase inhibitor leading to blockage of synthesis of thymidine, which eventually inhibits DNA replication.

Nucleoside analogues also present certain unwanted side effects such as nausea, vomiting, poor appetite, low blood counts.

Chemotherapeutics and apoptosis-inducing molecules, have been administered in the treatment of various forms of circulatory cancers such as leukemia and lymphoma, melanoma, bladder and kidney cancers, and other types of solid tumor cancers with a good degree of success. Cytokines may be characterized as protein molecules that play a role in regulating and directing the immune system. For cancer therapy, cytokines are synthesized and injected in larger does than the body normally produces. There are 2 common cytokines that are used in cancer immunotherapy, namely Interleukin 2 (IL2) and Interferon-α (IFN-α). In cancer treatment, IL2 is designed to target adaptive immune cells, T-cells and B-cells, to respond to tumors and IFN-α helps the body to generate innate immune cells, such as dendritic cells and macrophages that are designed to target the unhealthy cells.

TRAIL is a well-known cytotoxic protein which induces apoptosis in tumor cells, but not in normal cells. Tumors develop resistance to treatments with biologics such as TRAIL, like they do against other modalities of treatment such as chemotherapeutic agents. Therefore, although TRAIL is a highly desirable biologic that induces selective apoptosis in tumor cells, the tumors often develop resistance to TRAIL and thus their therapeutic utility is limited. Thus, there is an unmet need for providing a treatment which would include the administration of a new class of drug which would help overcome resistance to TRAIL and cause remission without undesirable side effects.

Chemotherapeutics, such as alkylating agents, act by attaching an alkyl group to the guanine base of DNA, thereby disrupting the normal functioning. They can be used on a wide range of cancers due to their ability to have this effect on a cancer cell on any phase of its life cycle. They can be used in the treatment of lymphoma, leukemia, testicular cancer, melanoma, brain cancer and breast cancer. Alkylating agents act by irreversibly binding to DNA. Once bound, the altered molecule leads to disruption of normal action and replication of DNA, ultimately leading to apoptosis or arrest of cellular replication. Therefore, upon treatment with alkylating agents, they can limit tumor growth and lead to tumor destruction. The most beneficial effect of alkylating agents is that it can be used to treat slow growing solid tumors and leukemia. They can also be used to treat lung cancer, ovarian cancer, breast cancer, lymphomas, sarcomas, myelomas, and Hodgkin's disease.

Representatives of alkylating agents can be classified as classical alkylating agents such as cyclophosphamide, mechlorethane; alkylating-like agents such as cisplatin, carboplatin; and agents such as procarbazine, ifosfamide, melphalan, chlorambucil, cyclophosphamide, streptozocin, carmustine, lomustine, busulfan, and dacarbazine. Mechlorethamine, marketed under the tradename MUSTARGEN®, is given by injection to treat Hodgkin's disease and non-Hodgkin's lymphoma, and as a palliative therapy for breast and lung cancers, and given as a topical treatment for skin lesions of mycosis fungoides (cutaneous T-cell lymphoma). Ifosfamide, sold under the tradename IFEX®, is used to treat both Hodgkin's and non-Hodgkin's lymphoma, as well as recurrent testicular cancer and germ cell tumors, sarcomas, lung cancer, bladder cancer, head and neck cancer, and cervical cancer. It is administered intravenously. Melphalan is a chemotherapy drug sold under the tradename ALKERAN®, and is also referred to as L-PAM or phenylalanine mustard. It is used to treat multiple myeloma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, and breast cancer. Chlorambucil is sold by the tradename LEUKERAN®, and is most widely used to treat chronic lymphocytic leukemia, malignant lymphomas including lymphosarcoma, giant follicular lymphoma, and Hodgkin's disease. It has also been successfully used to treat non-Hodgkin's lymphoma, breast, ovarian and testicular cancer, Waldenstrom's macroglobulinemia, thrombocythemia, and choriocarcinoma. It comes in coated tablet form. Cyclophosphamide is marketed under the tradename CYTOXAN® or NEOSAR®, and is used to treat Hodgkin's and non-Hodgkin's lymphoma, Burkitt's lymphoma, chronic lymphocytic leukemia, chronic myelocytic leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, t-cell lymphoma, multiple myeloma, neuroblastoma, retinoblastoma, rhabdomyosarcoma, Ewing's sarcoma and breast, testicular, endometrial, ovarian, and lung cancers. Streptozocin is sold under the tradename ZANOSAR®, and is used to treat islet cell pancreatic cancer. Carmustine is sold under the tradename BICNU®, and is used for some kinds of brain tumors, glioblastoma, brainstem glioma, medulloblastoma, astrocytoma, ependymoma, and metastatic brain tumors. It is also used in treatment for multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma, melanoma, lung cancer, and colon cancer. Lomustine, sold under the tradename CEENU®, is used to treat primary and metastatic brain tumors, Hodgkin's disease and non-Hodgkin's lymphoma, and has also been used for melanoma, lung, and colon cancer. Busulfan, sold under the tradenames BUSULFEX® and MYLERAN®, is used to treat chronic myelogenous leukemia. Dacarbazine is sold under the tradename DTIC-DOME® and is used to treat metastatic malignant melanoma, Hodgkin's disease, soft tissue sarcomas, neuroblastoma, fibrosarcomas, rhabdomyosarcoma, islet cell carcinoma, and medullary thyroid carcinoma. Temozolomide is sold under the tradename TEMODAR®, and is used to treat the specific types of brain tumors anaplastic astrocytoma and glioblastoma multiforme. Thiotepa, sold under the tradename THIOPLEX®, is an alkylating agent used to treat breast cancer, ovarian cancer, Hodgkin's disease, and non-Hodgkin's lymphoma. It is given by intravenous infusion. Altretamine is sold under the tradename HEXALEN® and is also called hexamethylmelamine or HMM. It is used to treat ovarian cancer.

Alkylating agents are also associated with side effects. They are toxic to normal cells as well, particularly rapidly dividing cells such as those in gastrointestinal tract and bone marrow. Long term use of alkylating agents can result in permanent infertility by reducing sperm production in males and leading to cessation of menstruation in females. Many alkylating agents can also lead to secondary cancers like acute myeloid leukemia.

Moreover, while the life expectancy under protein kinase inhibitor therapy, anthracycline therapy, nucleoside analog therapy, apoptosis-inducing molecular therapy or alkylating agent therapy is extended, it is not without undesirable side-effects effects of these drugs. Thus, there is an unmet need for providing a treatment which would include the administration of a new class of drug with a different mechanism of action, and which would result in a more complete remission profile without undesirable side effects.

SUMMARY OF THE INVENTION

This invention may be summarized inter alia to include:

A combination of antineoplastic agents useful for treating cancer comprising an effective amount of one or more nucleic acid molecules capable of down-regulating expression of at least one splice variant of the IG20 gene, wherein not all splice variants of the IG20 gene are down-regulated, and a chemotherapeutic selected from the group of one or more protein kinase inhibitor chemotherapeutics, one or more anthracycline chemotherapeutics, one or more nucleoside analogs, one or more apoptosis-inducing molecular therapeutics or one or more alkylating agent chemotherapeutics.

Such a combination, wherein the at least one splice variant of the IG20 gene is selected from a MADD splice variant, SNPs, allelic variations thereof, polymorphisms thereof, and genetic mutations thereof.

Such a combination, wherein the at least one splice variant of the IG20 gene is a MADD splice variant which exhibits exon 13L.

Such a combination, wherein the one or more nucleic acid molecules capable of down-regulating expression of the at least one splice variant of the IG20 gene is selected from siRNA, shRNA and antisense oligonucleotides.

Such a combination, wherein the siRNA, shRNA and antisense oligonucleotides comprise nucleic acids which are complementary to a nucleic acid sequence of exon 13L of a MADD splice variant, SNPs, allelic variations thereof, polymorphisms thereof, and genetic mutations thereof, and/or an mRNA transcript thereof.

Such a combination, wherein the nucleic acid molecule capable of down-regulating expression of at least one splice variant of the IG20 gene is comprised in an siRNA or shRNA.

Such a combination, wherein the siRNA and shRNA are encoded by a nucleic acid molecule which includes the structure:
Xsense−hairpin loop−Xanti-sense,
wherein X includes or consists essentially of a nucleic acid sequence CGGCGAATCTATGACAATC (SEQ ID NO:1).

Such a combination, wherein the siRNA or shRNA comprises a nucleic acid having the sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO:2).

Such a combination, wherein the siRNA or shRNA comprises a nucleic acid having the sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO:2) and is in the form of a duplex with a cognate nucleic acid having the sequence GAUUGUCAUAGAUUCGCCG (SEQ ID NO:10).

Such a combination, wherein the shRNA and siRNA are encoded by a nucleic acid having the sequence CGAATCTATGACAATCTTCAAGAGAGATTGTCATAGATTCGCCG (SEQ ID NO:3), wherein a hairpin loop region is from positions 20-28 of the sequence.

Such a combination, wherein the siRNA, shRNA and antisense oligonucleotide comprising nucleic acids which are complementary to a nucleic acid sequence of exon 13L of a MADD splice variant of the IG20 gene and/or an mRNA transcript thereof comprises a nucleic acid having the sequence selected from GAUUGUCAUAGAUUCGCCGTT (SEQ ID NO:4) and GAUUGUCAUAGAUUCGCCG (SEQ ID NO:10).

Such a combination, wherein the one or more nucleic acid molecules capable of down-regulating expression of the at least one splice variant of the IG20 gene is comprised in a drug delivery system.

Such a combination, wherein the drug delivery system is a targeted liposome formulation or a lentivirus vector.

Such a combination, wherein the one or more protein kinase inhibitor chemotherapeutics is selected from imatinib, trastuzumab, bevacizumab, gefitinib, cetuximab, Sorafenib, toceranib, erlotinib, lapatinib, sunitinib, nilotinib, bosutinib, neratinib and vatalanib and the cancer is selected from chronic myeloid leukemia, breast cancer, lung cancer, colorectal cancer, primary kidney cancer, liver cancer and thyroid cancer, and other types of solid tumor cancers as well as Kaposi's sarcoma.

Such a combination, wherein the one or more anthracycline chemotherapeutics is selected from daunorubicin, doxorubicin, epirubicin, idarubicin and valrubicin and the cancer is selected from leukemias, lymphomas, breast, stomach, uterine, ovarian, bladder cancer and lung cancers, and other types of solid tumor cancers as well as Kaposi's sarcoma.

Such a combination, wherein the one or more nucleoside analog chemotherapeutic is selected from gemcitabine, 5-fluorouracil and cytarabine and the cancer is selected from breast cancer, non-small cell lung cancer, pancreatic cancer, bladder cancer, biliary tract cancer and colon cancer, esophageal cancer, stomach cancer, cervical cancer, and other types of solid tumor cancers.

Such a combination, wherein the one or more apoptosis-inducing molecular therapeutic is selected from Interleukin 2 (IL2), Interferon-α (IFN-α) and Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) and the cancer is selected from circulatory cancers such as leukemia and lymphoma, melanoma, bladder and kidney cancers, and other types of solid tumor cancers.

Such a combination, wherein the one or more alkylating agent chemotherapeutic is selected cyclophosphamide, mechlorethane, cisplatin, carboplatin, procarbazine, ifosfamide, melphalan, chlorambucil, cyclophosphamide, streptozocin, carmustine, lomustine, busulfan, and dacarbazine and the cancer is selected from slow growing solid tumors and leukemia, lung cancer, ovarian cancer, breast cancer, lymphomas, sarcomas, myelomas, Hodgkin's disease, topical treatment for skin lesions of mycosis fungoides (cutaneous T-cell lymphoma), both Hodgkin's and non-Hodgkin's lymphoma, testicular cancer, germ cell tumors, bladder cancer, head and neck cancer, cervical cancer, multiple myeloma, neuroblastoma, rhabdomyosarcoma, lymphocytic leukemia, malignant lymphomas including lymphosarcoma, giant follicular lymphoma, Burkitt's lymphoma, chronic lymphocytic leukemia, chronic myelocytic leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, t-cell lymphoma, multiple myeloma, retinoblastoma, Ewing's sarcoma, endometrial cancer, islet cell pancreatic cancer, brain tumors, glioblastoma, brainstem glioma, medulloblastoma, astrocytoma, ependymoma, and metastatic brain tumors, colon cancer, fibrosarcomas, anaplastic astrocytoma, and glioblastoma multiforme.

Such a combination, wherein the one or more protein kinase inhibitor chemotherapeutic, one or more anthracycline chemotherapeutic, one or more nucleoside analog chemotherapeutic, one or more apoptosis-inducing molecular therapeutic, or one or more alkylating agent chemotherapeutic is in the form of a pharmaceutically acceptable salt.

Moreover, a method of treating cancers selected from chronic myeloid leukemia, breast cancer, lung cancer, colorectal cancer, primary kidney cancer, liver cancer and thyroid cancer, and other types of solid tumor cancers as well as Kaposi's sarcoma in a subject in need thereof, comprising administering an effective amount of a combination of antineoplastic agents comprising an effective amount of one or more nucleic acid molecules capable of down-regulating expression of at least one splice variant of the IG20 gene, wherein not all splice variants of the IG20 gene are down-regulated, and one or more protein kinase inhibitor chemotherapeutic.

Such a method, wherein the one or more protein kinase inhibitor chemotherapeutic is selected from imatinib, trastuzumab, bevacizumab, gefitinib, cetuximab, sorafenib, toceranib, erlotinib, lapatinib, sunitinib, nilotinib, bosutinib, neratinib and vatalanib.

Moreover, a method of treating cancers selected from leukemias, lymphomas, breast, stomach, pancreas, uterine, ovarian, cervical, prostate, melanoma, esophageal, bladder cancer, liver cancer and thyroid tumors, and lung cancers, and other types of solid tumor cancers as well as Kaposi's sarcoma in a subject in need thereof, comprising administering an effective amount of a combination of antineoplastic agents comprising an effective amount of one or more nucleic acid molecules capable of down-regulating expression of at least one splice variant of the IG20 gene, wherein not all splice variants of the IG20 gene are down-regulated, and one or more anthracycline chemotherapeutic.

Such a method, wherein the one or more anthracycline chemotherapeutic is selected from daunorubicin, doxorubicin, epirubicin, idarubicin and valrubicin.

Moreover, a method of treating cancers selected from breast cancer, non-small cell lung cancer, pancreatic cancer, bladder cancer, biliary tract cancer and colon cancer, esophageal cancer, stomach cancer, pancreatic cancer, breast cancer, cervical cancer, and other types of solid tumor cancers in a subject in need thereof, comprising administering an effective amount of a combination of antineoplastic agents comprising an effective amount of one or more nucleic acid molecules capable of down-regulating expression of at least one splice variant of the IG20 gene, wherein not all splice variants of the IG20 gene are down-regulated, and one or more nucleoside analog chemotherapeutic.

Such a method, wherein the one or more nucleoside analog chemotherapeutic is selected from gemcitabine, 5-fluorouracil and cytarabine.

Moreover, a method of treating cancers selected from circulatory cancers such as leukemia and lymphoma, melanoma, bladder and kidney cancers, and other types of solid tumor cancers in a subject in need thereof, comprising administering an effective amount of a combination of antineoplastic agents comprising an effective amount of one or more nucleic acid molecules capable of down-regulating expression of at least one splice variant of the IG20 gene, wherein not all splice variants of the IG20 gene are down-regulated, and one or more apoptosis-inducing molecular therapeutic.

Such a method, wherein the one or more apoptosis-inducing molecular therapeutic is selected from Interleukin 2 (IL2), Interferon-α (IFN-α) and Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL).

Moreover, a method of treating cancers selected from slow growing solid tumors and leukemia, lung cancer, ovarian cancer, breast cancer, lymphomas, sarcomas, myelomas, Hodgkin's disease, topical treatment for skin lesions of mycosis fungoides (cutaneous T-cell lymphoma), both Hodgkin's and non-Hodgkin's lymphoma, testicular cancer, germ cell tumors, bladder cancer, head and neck cancer, cervical cancer, multiple myeloma, neuroblastoma, rhabdomyosarcoma, lymphocytic leukemia, malignant lymphomas including lymphosarcoma, giant follicular lymphoma, Burkitt's lymphoma, chronic lymphocytic leukemia, chronic myelocytic leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, t-cell lymphoma, multiple myeloma, retinoblastoma, Ewing's sarcoma, endometrial cancer, islet cell pancreatic cancer, brain tumors, glioblastoma, brainstem glioma, medulloblastoma, astrocytoma, ependymoma, and metastatic brain tumors, colon cancer, fibrosarcomas, anaplastic astrocytoma, and glioblastoma multiforme in a subject in need thereof, comprising administering an effective amount of a combination of antineoplastic agents comprising an effective amount of one or more nucleic acid molecules capable of down-regulating expression of at least one splice variant of the IG20 gene, wherein not all splice variants of the IG20 gene are down-regulated, and one or more alkylating agent chemotherapeutic.

Such a method, wherein the one or more alkylating agent chemotherapeutic is selected from cyclophosphamide, mechlorethane, cisplatin, carboplatin, procarbazine, ifosfamide, melphalan, chlorambucil, cyclophosphamide, streptozocin, carmustine, lomustine, busulfan, and dacarbazine.

Such methods of treating cancers, wherein the cancers exhibit expression of at least one splice variant of the IG20 gene.

Such methods of treating cancers, wherein the cancers exhibit expression of the MADD splice variant of the IG20 gene, SNPs, allelic variations thereof, polymorphisms thereof, and genetic mutations thereof.

Such methods of treating cancers, wherein MADD splice variant exhibits exon 13L of the IG20 gene.

Such methods of treating cancers, wherein the one or more nucleic acid molecules capable of down-regulating expression of at least one splice variant of the IG20 gene is selected from siRNA, shRNA and antisense oligonucleotides.

Such methods of treating cancers, wherein the siRNA, shRNA and antisense oligonucleotides comprise nucleic acids which are complementary to a nucleic acid sequence of exon 13L of a MADD splice variant, SNPs, allelic variations thereof, polymorphisms thereof, and genetic mutations thereof, of the IG20 gene and/or an mRNA transcript thereof.

Such methods of treating cancers, wherein the nucleic acid molecule capable of down-regulating expression of at least one splice variant of the IG20 gene is comprised in siRNA or shRNA.

Such methods of treating cancers, wherein the siRNA and shRNA is encoded by a nucleic acid molecule which includes the structure:
Xsense−hairpin loop−Xanti-sense,
wherein X includes or consists essentially of a nucleic acid sequence CGGCGAATCTATGACAATC (SEQ ID NO:1).

Such methods of treating cancers, wherein the siRNA and shRNA comprises a nucleic acid having the sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO:2).

Such methods of treating cancers, wherein the siRNA and shRNA comprises a nucleic acid having the sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO:2) and is in the form of a duplex with a cognate nucleic acid having the sequence GAUUGUCAUAGAUUCGCCG (SEQ ID NO:10).

Such methods of treating cancers, wherein the shRNA is encoded by a nucleic acid having the sequence CGAATCTATGACAATCTTCAAGAGAGATTGTCATAGATTCGCCG (SEQ ID NO:3), wherein a hairpin loop region is from positions 20-28 of the sequence.

Such methods of treating cancers, wherein the siRNA, shRNA and antisense oligonucleotides comprising nucleic acids which are complementary to a nucleic acid sequence of exon 13L of a MADD splice variant of the IG20 gene and/or an mRNA transcript thereof comprise a nucleic acid having the sequence selected from GAUUGUCAUAGAUUCGCCGTT (SEQ ID NO:4) and GAUUGUCAUAGAUUCGCCG (SEQ ID NO:10).

Such methods of treating cancers, wherein the one or more siRNA, shRNA and antisense oligonucleotides is administered in the form of a liposomal formulation or by lentivirus transfection.

Such methods of treating cancers, wherein the one or more siRNA, shRNA and antisense oligonucleotides is administered as an adjuvant.

Such methods of treating cancers, wherein the one or more protein kinase inhibitor chemotherapeutic, one or more anthracycline chemotherapeutic, one or more nucleoside analog chemotherapeutic, one or more apoptosis-inducing molecular therapeutic or the one or more alkylating agent chemotherapeutic is administered in the form of a pharmaceutical composition further comprising one or more pharmaceutically acceptable diluents, excipients, or carriers.

Such methods of treating cancers, wherein the one or more siRNA, shRNA and antisense oligonucleotides is administered prior to the one or more protein kinase inhibitor chemotherapeutic, one or more anthracycline chemotherapeutic, one or more nucleoside analog chemotherapeutic, one or more apoptosis-inducing molecular therapeutic or the one or more alkylating agent chemotherapeutic; or is administered simultaneously with the one or more protein kinase inhibitor chemotherapeutic, one or more anthracycline chemotherapeutic, one or more nucleoside analog chemotherapeutic, one or more apoptosis-inducing molecular therapeutic or the one or more alkylating agent chemotherapeutic.

Such methods of treating cancers, wherein the one or more siRNA, shRNA and antisense oligonucleotides is administered in the form of a pharmaceutical composition further comprising one or more pharmaceutically acceptable diluents, excipients, or carriers.

Such methods of treating cancers, wherein the one or more siRNA, shRNA, and antisense oligonucleotides and the one or more protein kinase inhibitor chemotherapeutic, one or more anthracycline chemotherapeutic, one or more nucleoside analog chemotherapeutic, one or more apoptosis-inducing molecular therapeutic or the one or more alkylating agent chemotherapeutic are formulated in a dosage pack and are administered according to a selected treatment regime.

