WO2018129622A1 - Dual targeting antisense oligonucleotides for use as apoptotic inhibitors for the treatment of cancer - Google Patents

Abstract

Provided herein are compositions, method and uses for modulating IAP activity or for the treatment of cancer. The compositions comprise dual-targeting antisense oligonucleotides (dASO) for administration to a cancer cell, wherein the cancer cell may be characterized by elevated expression of one of more of BIRC6 or cIA1. The cancer may be selected from one or more of: prostate cancer; childhood de novo acute myeloid leukemia; colorectal cancer; neuroblastoma; melanoma; liver cancer; non-small cell lung cancer; epithelial ovarian cancer; hepatocellular carcinoma; esophageal squamous cell carcinoma; drug-resistant chronic myelogenous leukemia (CML); and breast cancer. The prostate cancer may be castration-resistant prostate cancer (CRPC).

Description

DUAL TARGETING ANTISENSE OLIGONUCLEOTIDES FOR USE AS APOPTOTIC INHIBITORS FOR THE TREATMENT OF CANCER

TECHNICAL FIELD

[0001] The present invention provides compounds, compositions and methods for modulating the expression of human Inhibitors of Apoptosis (IAPs). In particular, this invention relates to dual-targeting antisense oligonucleotides (dASOs) capable of modulating human BIRC6 and/or cIAPi mRNA expression, and their uses and methods for the treatment of various indications, including various cancers. In particular, the invention relates to therapies and methods of treatment for cancers such as prostate cancer, including castration-resistant prostate cancer (CRPC).

BACKGROUND

[0002] Prostate cancer is the most common non-cutaneous cancer and the second leading cause of cancer-related deaths for males in the Western world (Siegel R, et ah, 2012., 62(i):io- 29). Prostate cancers are initially androgen-dependent, and while androgen deprivation therapy (ADT) can induce marked tumor regression, resistance to ADT inevitably emerges, leading to castration-resistant prostate cancer (CRPC). The current standard care for treating CRPC is systemic, docetaxel-based chemotherapy, increasing the overall survival of patients by about 2 months compared to mitoxantrone-based therapy (Petrylak DP, et ah, N Engl J Med. 2004; 351(15): 1513-1520; Tannock IF, et ah, N Engl J Med. 2004; 351(15): 1502-1512). Recently, sipuleucel-T, cabazitaxel, abiraterone, MDV3100 and Radium-223 have shown more prolonged overall survival benefit and are approved by the FDA for treatment of the disease (Bishr M and Saad F., Nat Rev Urol. 2013; 10(9)1522-528). However, none of these drugs are curative; they incrementally improve overall survival. The establishment of more effective therapeutic targets and drugs, specifically those targeting the molecular drivers of metastatic CRPC, is of critical importance for improved disease management and patient survival (Lin D, et ah, Curr Opin Urol. 2013; 23(3):214-219).

[0003] Apoptosis, a cell death-inducing process important in the regulation of cell numbers in normal tissues, can be triggered by a variety of death signals from both extracellular and intracellular origins, and involves activation of caspases (intracellular cysteine proteases) that mediate the execution of apoptosis (Hensley P, et ah, Biol Chem. 2013; 394(7):83i-843). Human cancers are characterized by resistance to apoptosis, intrinsic or acquired, considered to be a key factor underlying resistance to therapeutic intervention, and promising new strategies have been developed based on drug-induced apoptosis (Gleave M, et al., Cancer Chemother Pharmacol. 2005; 56 Suppl 1:47-57). The treatment resistance of CRPC is thought to be based on an increased resistance to apoptosis by the prostate cancer cells and may be addressed by targeting anti- apoptotic genes and their products (Zielinski RR, et al, Cancer J. 2013; i9(i):79-89).

[0004] The Inhibitors of Apoptosis (IAP) form a family of functionally and structurally related proteins that have a major role in cell death regulation. They act as endogenous apoptosis inhibitors by binding to caspases, thereby suppressing apoptosis initiation. The human IAP family consists of 8 members that are characterized by the presence of 1 to 3 baculovirus inhibitor of apoptosis repeat (BIR) motifs that are involved in the binding of IAPs to caspases. There is increasing evidence that IAPs also affect other cellular processes, such as ubiquitin-dependent signalling events that activate nuclear factor κΒ (NFKB) transcription factors, which in turn drive the expression of genes important in cellular processes such as cell survival (Gyrd-Hansen M and Meier P. Nat Rev Cancer. 2010; io(8):56i-574). Due to their ability to control cell death and elevated expression in a variety of cancer cell types, IAP proteins are attractive targets for the development of novel anti-cancer treatments (de Almagro MC and Vucic D. Exp Oncol. 2012; 34(3):200-2ii). Four LAP members, i.e. XIAP, survivin, cIAPi and CIAP2, have been reported to be up-regulated in prostate cancer (Krajewska M, et ah, Clin Cancer Res. 2003; 9(i3):49i4-4925).

[0005] The BIRC6 gene (BRUCE/APOLLON) encodes a 528 kDa protein in mammals, consisting of a single N-terminal BIR domain and a C-terminal ubiquitin-conjugating (UBC) domain; the latter has chimeric E2/E3 ubiquitin ligase activity as well as anti-apoptotic activity (Bartke T, et ah, Mol Cell. 2004; i4(6):8oi-8n). Through its BIR domain, BIRC6 protein can bind to active caspases, including caspases-3, 6, 7 and 9 and such interactions have been shown to underlie its ability to inhibit the caspase cascade and ultimately apoptosis (Bartke T, et ah, Mol Cell. 2004; 14(6):8OI-8II). Through its UBC domain, BIRC6 facilitates proteasomal degradation of pro-apoptotic proteins, including caspase-9 (Hao Y, et ah, Nat Cell Biol. 2004; 6(9):849-86o), SMAC/DIABLO (Hao Y, et al., Nat Cell Biol. 2004; 6(9):849"86o; Qiu XB and Goldberg AL. J Biol Chem. 2005; 28o(i):i74-i82), and HTRA2/OMI (Bartke T, et ah, Mol Cell. 2004; i4(6):8oi-8n; Sekine K, et al., Biochem Biophys Res Commun. 2005; 33θ(ι):279-285). Elevated expression of BIRC6 has been found in a variety of cancers, i.e. childhood de novo acute myeloid leukemia (Sung KW, et ah, Clin Cancer Res. 2007; I3(i7):5i09-5ii4), colorectal cancer (Bianchini M, et ah, Int J Oncol. 2006; 29(i):83-94), neuroblastoma (Bartke T, et ah, Mol Cell. 2004; i4(6):8oi-8n; Lamers F, et ah, BMC Cancer. 2012; 12:285), melanoma (Tassi E, et ah, Clin Cancer Res. 2012; i8(i2):33i6-3327) and non-small cell lung cancer (Dong X, etal., J Thorac Oncol. 2013; 8(2):i6i- 170). Furthermore, BIRC6 has been implicated in maintaining resistance against cell death stimuli [Chen Z, et al., Biochem Biophys Res Commun. 1999; 204(3):847-854; Chu L, et al., Gene Ther. 2008; 15(7):484"494). In contrast to other IAPs, BIRC6 has been shown to have a cytoprotective role, essential for survival of mammalian cells (Hao Y, et al, Nat Cell Biol. 2004; 6(9):849-86o; Qiu XB, et al, EMBO J. 2004; 23(4):8oo-8io). BIRC6 is also known for its essential role in regulating cytokinesis, a final event of cell division (Pohl C and Jentsch S. Cell. 2008; i32(5):832-845). The dual roles of BIRC6 in cell death and division processes resemble those of survivin, and render it a promising target for therapy of a variety of cancers (Martin SJ. Nat Cell Biol. 2004; 6(9):8θ4-8θ6).

[0006] Therapeutic options for castration resistant prostate cancer (CRPC) treatment have changed considerably with the recent FDA approvals of newer agents that improve patient survival. In particular, Enzalutamide (ENZ), a second generation androgen receptor antagonist approved for treating metastatic CRPC in post -docetaxel and more recently, pre-docetaxel setting. However, within 2 years of clinical practice, development of ENZ resistance was evident in majority of patients (Claessens et al. 2014) and no known therapies were shown to be effective to ENZ-resistant CRPC to-date. Thus, a novel therapeutic agent that can effectively suppress ENZ-resistant CRPC would be useful.

SUMMARY

The present invention is based in part on the discovery that the inhibition of BIRC6 expression and the inhibition of cIAPi expression with dual -targeting antisense oligonucleotide (dASO) (which may be selected from any one or more of SEQ ID NOs:i-4, 7-9, 11, 15, 17 or 20) may be useful in the treatment of cancer. Similarly, the invention is also based in part of the discovery that the inhibition of BIRC6 expression may be useful in the treatment of cancer. Furthermore, that a reduction in proliferation of cancer cells resulting from the administration of some dual- targeting antisense oligonucleotides may extend to castration-resistant prostate cancer (CRPC).

[0007] In a first aspect, a composition is provided a method for the treatment of a cancer cell, the method including administering an oligonucleotide of one or more of SEQ ID NOs:i-4, 7-9, 11, 15, 17 or 20 to the cell. In a further aspect, a composition is provided a method for the treatment of a cancer cell, the method including administering an oligonucleotide of one or more of SEQ ID NOs:i-4, 7, 8, 11 or 15 to the cell. In a further aspect, a composition is provided a method for the treatment of a cancer cell, the method including administering an oligonucleotide of one or more of SEQ ID NOs:i-4 or 11 to the cell. In a further aspect, a composition is provided a method for the treatment of a cancer cell, the method including administering an oligonucleotide of one or more of SEQ ID NOs:i-4 to the cell. [0008] In a further aspect, there is provided a method including administering a dual- targeting antisense oligonucleotides (dASO) of any one of SEQ ID NOs:i-4, 7-9, 11, 15, 17 or 20. In a further aspect, there is provided a method including administering a dual-targeting antisense oligonucleotides (dASO) of any one of SEQ ID NOs:i-4, 7, 8, 11 or 15. In a further aspect, there is provided a method including administering a dual-targeting antisense oligonucleotides (dASO) of any one of SEQ ID NOs:i-4 or 11. In a further aspect, there is provided a method including administering a dual -targeting antisense oligonucleotides (dASO) of any one of SEQ ID NOs:i-4.

[0009] In a further aspect, there is provided a dual-targeting antisense oligonucleotide (dASO), wherein the oligonucleotide has a sequence selected from one or more of SEQ ID NOs:i-4, 7-9, 11, 15, 17 or 20. In a further aspect, there is provided a dual-targeting antisense oligonucleotide (dASO), wherein the oligonucleotide has a sequence selected from one or more of SEQ ID NOs:i-4, 7, 8, 11 or 15. In a further aspect, there is provided a dual -targeting antisense oligonucleotide (dASO), wherein the oligonucleotide has a sequence selected from one or more of SEQ ID NOs:i-4 or 11. In a further aspect, there is provided a dual-targeting antisense oligonucleotide (dASO), wherein the oligonucleotide has a sequence selected from one or more of SEQ ID NOs:i-4.

[0010] In a further aspect, there is provided a dual-targeting antisense oligonucleotide (dASO) selected from one or more of SEQ ID NOs: 1-4, 7-9, 11, 15, 17 or 20 for use in the treatment of cancer. In a further aspect, there is provided a dual-targeting antisense

oligonucleotide (dASO) selected from one or more of SEQ ID NOs: 1-4, 7, 8, 11 or 15 for use in the treatment of cancer. In a further aspect, there is provided a dual-targeting antisense oligonucleotide (dASO) selected from one or more of SEQ ID NOs: 1-4, or 11 for use in the treatment of cancer. In a further aspect, there is provided a dual-targeting antisense

oligonucleotide (dASO) selected from one or more of SEQ ID NOs: 1-4 for use in the treatment of cancer.

[0011] In a further aspect, there is provided a dual-targeting antisense oligonucleotide (dASO) of SEQ ID NO:i-4, 7-9, 11, 15, 17 or 20 for inhibiting expression of one of more of BIRC6 and cIAPi. In a further aspect, there is provided a dual-targeting antisense oligonucleotide (dASO) of SEQ ID NO: 1-4, 7, 8, 11 or 15 for inhibiting expression of one of more of BIRC6 and cIAPi. In a further aspect, there is provided a dual-targeting antisense oligonucleotide (dASO) of SEQ ID NO:i-4 or 11 for inhibiting expression of one of more of BIRC6 and cIAPi. In a further aspect, there is provided a dual-targeting antisense oligonucleotide (dASO) of SEQ ID NO: 1-4 for inhibiting expression of one of more of BIRC6 and cIAPi. [0012] In a further aspect, there is provided a pharmaceutical composition, the composition including (a) dual-targeting antisense oligonucleotide (dASO) selected from one or more of SEQ

ID NO: 1-4, 7-9, 11, 15, 17 or 20; and (b) a pharmaceutically acceptable carrier.

[0013] In a further aspect, there is provided a use of a dASO selected from one or more of

SEQ ID NO: 1-4, 7-9, 11, 15, 17 or 20 in the preparation of a medicament for the treatment of cancer.

[0014] In a further aspect, there is provided a use of a dASO selected from one or more of SEQ ID NO: 1-4, 7-9, 11, 15, 17 or 20 in the preparation of a medicament for inhibiting expression of one of more of BIRC6 and cIAPi.

[0015] In a further aspect, there is provided a use of a dASO selected from one or more of SEQ ID NO: 1-4, 7-9, 11, 15, 17 or 20 for the treatment of cancer.

[0016] In a further aspect, there is provided a use of a dASO selected from one or more of SEQ ID NO: 1-4, 7-9, 11, 15, 17 or 20 for inhibiting expression of one of more of BIRC6 and cIAPi.

[0017] In accordance with a further aspect, there is provided a commercial package including (a) an antisense oligonucleotide sequence described herein and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for modulating LAP activity.

[0018] In a further aspect, there is provided a commercial package, including: (a) dASO selected from one or more of SEQ ID NO: 1-4, 7-9, 11, 15, 17 or 20; and (b) instructions for the treatment of cancer.

[0019] In accordance with a further aspect, there is provided a method for modulating LAP activity, the method comprising administering a dual-targeting antisense oligonucleotides (dASO) selected from one or more of SEQ ID NO: 1-4, 7-9, 11, 15, 17 or 20 and a

pharmaceutically acceptable carrier; and (b) instructions for the use thereof.

[0020] In accordance with a further aspect, there is provided a method for modulating LAP activity in a cell, the method comprising administering a dual-targeting antisense

oligonucleotides (dASO) selected from one or more of SEQ ID NO: 1-4, 7-9, 11, 15, 17 or 20 to the cell.

[0021] In accordance with a further aspect, there is provided a use of a dual-targeting antisense oligonucleotides (dASO) selected from one or more of SEQ ID NO: 1-4, 7-9, 11, 15, 17 or 20 for modulating LAP activity.

[0022] In accordance with a further aspect, there is provided a use of a dual-targeting antisense oligonucleotides (dASO) selected from one or more of SEQ ID NO: 1-4, 7-9, 11, 15, 17 or 20 in the manufacture of a medicament for modulating LAP activity. [0023] The dASO may further include a modified internucleoside linkage. The modified internucleoside linkage may be a peptide-nucleic acid linkage, a morpholino linkage, a N3' to P5' phosphoramidate linkage, a methylphosphonate linkage or a phosphorothioate linkage. The dASO may further include a modified sugar moiety. The modified sugar moiety may be a 2 ' -O- alkyl oligoribonucleotide. The dASO may further have a 2'MOE gapmer modification. The dASO may further include a modified nucleobase. The modified nucleobase may be a 5-methyl pyrimidine or a 5- propynyl pyrimidine.

[0024] The cell may be a human cell. The cancer may be characterized by elevated expression of one of more of BIRC6 and cIAPi. The cancer may be selected from one or more of the following: prostate cancer; childhood de novo acute myeloid leukemia; colorectal cancer; neuroblastoma; melanoma; liver cancer; non-small cell lung cancer; epithelial ovarian cancer; hepatocellular carcinoma; esophageal squamous cell carcinoma; drug-resistant chronic myelogenous leukemia (CML); and breast cancer. The prostate cancer maybe castration- resistant prostate cancer (CRPC). The prostate cancer may be enzalutamide (ENZ) resistant CRPC. Furthermore, the dASOs may also be useful in the treatment of Myelodysplastic syndromes and Pseudoexfoliative glaucoma.

[0025] The dASO may be substantially complementary to the mRNA of BIRC6 and cIAPi. The dASO may be administered intranvenously. The dASO may be topically administered to a tissue. The dASO may be mixed with lipid particles prior to administration. The dASO may be encapsulated in liposomes prior to administration.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIGURE 1 shows BIRC6 mRNA expression of PC-3 cells transfected with various 6w2 length variants (Gen 1.0), where the results represent mean relative quantity of duplicate experiments (Error bars indicate SD).

[0027] FIGURES 2A and 2B show the effect of Gen2.5 dASO-6w2 variants on BIRC6 (A) and cIAPi (B) mRNA expression in PC3 cells at various concentrations of dASO.

[0028] FIGURES 3A and 3B show the effect of Gen2.5 dASO-6w2 variants on BIRC6 and cIAPi protein expression in PC3 cells at various concentrations of dASO.

[0029] FIGURE 4 shows that Gen2.5 dASO-6w2 variants significantly suppress proliferation of PC-3 prostate cancer cells in vitro.

[0030] FIGURE 5A-J show Gen2.5 ASO-6w2-A suppresses Tumour growth in multiple prostate cancer PDX models, wherein A-F, Tumour growth curve of 6 PDX lines grafted subcutaneously, and wherein A-C, hormone naive PCa. D-E, CRPC. F, NEPC. G-J, Serum PSA curves and tumour volume at harvest of PDX lines grafted at subrenal capsule site. G-H, LTL- 412 hormone naive PCa. I-J, LTL-555, hormone naive PCa.

[0031 ] FIGURE 6 shows Treated/Control (T/C) tumour volume ratios (6w2-A/Ctrl), where the ratio is the tumour volume in PDX tumour lines, and wherein the T/ C ratio < 1 indicates growth suppression by the ASO (smaller values indicate greater growth suppression).

[0032] FIGURE 7 shows mouse body weights in four PDX lines, wherein the results are presented as ratios of individual mouse body weight at treatment end day/ first day.

[0033] FIGURE 8 shows IHC staining of cleaved caspase-3 showing increased apoptosis in tumours of mice treated with Gen2.5 dASO-6w2-A, wherein the dark staining indicates cleaved caspase-3 protein expression.

[0034] FIGURE 9 shows H & E staining showing defective tumour vasculature and blood pooling (arrows) in Gen2.5 dASO-6w2-A-treated mice in three PDX lines.

[0035] FIGURE 10 shows the expression of BIRC6 and cIAPi genes in PDX tumours harvested at the end of treatment, wherein Gen2.5 6w2-A inhibits tumour BIRC6 and cIAPi mPvNA in LTL-573R and BIRC6 in LTL-484.

DETAILED DESCRIPTION

[0036] Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the present field of art. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions, devices, methods and the like of embodiments, and how to make or use them. It will be appreciated that the same thing may be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. No significance is to be placed upon whether or not a term is elaborated or discussed herein. Some synonyms or substitutable methods, materials and the like are provided. Recital of one or a few synonyms or equivalents does not exclude use of other synonyms or equivalents, unless it is explicitly stated. Use of examples in the specification, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the embodiments described herein.

[0037] A method is provided for "treating" a cancer cell, wherein treating is meant to encompass preventing proliferation of the cell, ameliorating symptoms associated with the cancer, and eradicating the cancer cell. The term "treating" as used herein is also meant to include the administration at any stage of the cancer, including early administration of a compound or late administration. A person of skill in the art would appreciate that the term "ameliorating" is meant to include the prospect of making a cancer more tolerable for a subject afflicted therewith (for example, by reducing tumour load). A person of skill in the art would also appreciate that the term "eradication" with regards to cancer would include elimination of the cancer cells in whole or in part from a subject. Accordingly, as used herein "treatment" may refer to the prevention of cancer cell proliferation in a subject, the amelioration of symptoms associated with the cancer, the eradication of the cancer from a subject, or combinations thereof.

[0038] Antisense oligonucleotide compounds are typically single stranded RNA compounds which bind to complementary RNA compounds, such as target mRNA molecules, and block translation from the complementary RNA compounds by sterically interfering with the normal translational machinery. This process is usually passive, in that it does not require or involve additional enzymes to mediate the RNA interference process. Specific targeting of antisense RNA compounds to inhibit the expression of a desired gene may generally involve designing the antisense RNA compound to have a homologous, complementary sequence to the desired gene. Perfect homology is not necessary for the RNA interference effect. In one embodiment of the invention, the antisense RNA compounds include any RNA compound with sufficient complementary homology to bind to the BIRC6 mRNA transcript and the cIAPi transcript causing a reduction in translation of the BIRC6 and cIAPi proteins. The antisense compounds be modified to enhance the stability of the oligonucleotides, particularly for in vivo use. Numerous examples of methods for designing and optimizing antisense RNA compounds are found in the journal literature - i.e. (Pan and Clawson 2006; Patzel 2007; Peek and Behlke 2007). Perfect sequence complementarity is not necessary for the antisense compound to modulate expression of the target gene. The present inventors provide non-limiting examples of antisense compounds which modulate the expression of BIRC6 and cIAPi.

[0039] Antisense oligonucleotide sequences as described herein or for use as described herein may be administered by means of a medical device or appliance such as an implant, graft, prosthesis, stent, etc. Also, implants may be devised which are intended to contain and release such compounds or compositions. An example would be an implant made of a polymeric material adapted to release the compound over a period of time.

[0040] Dual-targeting antisense oligonucleotides (dASOs) that simultaneously target BIRC6 and another co-upregulated LAP member are provided herein. Encompassed herein, is the use of dASOs derived from lcjmer 6w2 for administration to a cell. The 6w2 dASOs as used in the examples had a modified internucleoside linkages. In particular, the dASOs had phosphorothioate linkages between all nucleosides. 6w2 derived dASOs (for example, SEQ ID NO:i-4, 7-9, 11, 15, 17 and 20) may have combined inhibition of BIRC6 expression and cIAPi expression. 6w2 derivatives SEQ ID NO: 1-4, 7-9, 11, 15, 17 and 20 may be oligonucleotides with modified phosphorothioate linkages. However, variations of 6w2 derivatives SEQ ID NO: 1-4, 7- 9, 11, 15, 17 and 20 may also have unmodified phosphodiester linkages or partially modified linkages (i.e. any integer between 1 and 18 phosphorothioate linkage(s) or other modified linkages). Alternative modifications are also known in the art.

[0041] A phosphorothioate oligonucleotide bond modification alters the phosphate linkage by replacing one of the non-bridging oxygens with sulfur. The introduction of phosphorothioate linkages alters the chemical properties of the oligonucleotide. In particular, the addition of phosphorothioate linkages reduces nuclease degradation of the oligonucleotide and thereby increasing the half-life in situ. Accordingly, this modification is particularly useful for antisense oligonucleotides, which when introduced into cells or biological matrices can interact with target nucleic acids to silence the expression of a particular transcript. Oligonucleotides containing phosphorothioate linkages accomplish this feat either through direct blockage of translation or enable enzymatic degradation of the target transcript (for example, via RNase H). Gen 2.5 6w2 delta A (SEQ ID NO:i), Gen 2.5 6w2 delta B (SEQ ID N0:2), Gen 2.5 6w2 delta C (SEQ ID NO:3) and Gen 2.5 6w2 delta D (SEQ ID NO:4) are 3-1—3 (S)-cEt gapmers with phosphorothioate backbones.

[0042] Although phosphorothioate linkages provide improved half-life, the introduction of these linkages into an oligonucleotide may also introduce limitations to their function as antisense oligonucleotides. Each phosphorothioate linkage creates a chiral center at each bond, which may result in multiple isomers of the oligonucleotide generated during synthesis and the isomers may have differential characteristics and functional properties. However much of the isomer effects may be mitigated through careful positioning of the modifications or by using additional modifications in conjunction with the phosphorothioate bonds.

[0043] One or more of the phosphorothiodiester linkages of the oligonucleotide moiety may be modified by replacing one or both of the two bridging oxygen atoms of the linkage with analogues such as -NH, -CH2, or -S. Other oxygen analogues known in the art may also be used.

[0044] A "modified oligonucleotide" as used herein is meant to include oligonucleotides that are substituted or modified. In addition to the naturally occurring primary bases adenine, guanine, cytosine, and thymine, or other natural bases such as inosine, deoxyinosine, and hypoxanthine, there are numerous other modifications. For example, isosteric purine 2 ' deoxy- furanoside analogues, 2 ' -deoxynebularine or 2 ' deoxyxanthosine, or other purine and pyrimidine analogues such as 5-methyl pyrimidine or a 5-propynyl pyrimidine may also be utilized to improve stability and target hybridization. [0045] A "modified sugar" as used herein when discussing an oligonucleotide moiety, a sugar modified or replaced so as to be ribose, glucose, sucrose, or galactose, or any other sugar. Alternatively, the oligonucleotide may have one or more of its sugars substituted or modified in its 2' position, i.e. 2 ' alkyl or 2'-o-alkyl. An example of a 2 ' -O-allyl sugar is a 2 ' -O- methylribonucleotide. Furthermore, the oligonucleotide may have one or more of its sugars substituted or modified to form an a-anomeric sugar. Newer bicyclic nucleic acid (BNA) modifications may also be used (for example, BNA including constrained ethyl (cEt) and/or locked nucleic acid (LNA)). cET and LNA may be regarded as "modified sugar moiety".

[0046] "Second-generation" oligonucleotides as used herein mat be defined as oligonucleotides that are resistant to degradation by cellular nucleases and capable of hybridizing specifically to their target mRNA with equal or higher affinity than first generation ASOs. An example of a 2^nd generation ASO is a 2'-0-(2-Methoxyethyl)-RNA (2'MOE gapmer modification). With a 2'-M0E gapmer the 5' and 3' ends may have 2'-M0E modified nucleotides to protect against degradation, but the gap between the 5' and 3' ends may be unmodified phosphodiester linkages. Numerous other chemical modifications have been developed to improve ASOs. For example, morpholino, N3' to P5' phosphoramidate, and methylphosphonate chemical modifications are known in the art (N. Dias, and C. A. Stein 2002). Furthermore, peptide nucleic acids (PNAs) may also be used.

[0047] 6w2 Sequence Length variants are shown below in TABLE A.

[0048] TABLE A: Sequence Length Variants for 6w2 dASOs.

14nt-vl4 tgcagcatcatgtg Y 20

[0049] The compounds, as described herein, may be in isolation, or may be linked to or in combination with tracer compounds, liposomes, carbohydrate carriers, polymeric carriers or other agents or excipients as will be apparent to one of skill in the art. In alternate embodiments, such compounds may further comprise an additional medicament, wherein such compounds may be present in a pharmacologically effective amount.

[0050] The term "medicament" as used herein refers to a composition that may be administered to a patient or test subject and is capable of producing an effect in the patient or test subject. The effect may be chemical, biological or physical, and the patient or test subject may be human, or a non-human animal, such as a rodent (for example, a transgenic mouse, a mouse or a rat), dog, cat, cow, sheep, horse, hamster, guinea pig, rabbit or pig. The medicament may be comprised of the effective chemical entity alone or in combination with a pharmaceutically acceptable excipient.

[0051] The term "pharmaceutically acceptable excipient" may include any and all solvents, dispersion media, coatings, antibacterial, antimicrobial or antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. An excipient may be suitable for intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, topical or oral administration. An excipient may include sterile aqueous solutions or dispersions for extemporaneous preparation of sterile injectable solutions or dispersion. Use of such media for preparation of medicaments is known in the art.

[0052] Compositions or compounds according to some embodiments described herein may be administered in any of a variety of known routes. Examples of methods that may be suitable for the administration of a compound include orally, intravenous, inhalation, intramuscular, subcutaneous, topical, intraperitoneal, intra-rectal or intra-vaginal suppository, sublingual, and the like. The compounds described herein may be administered as a sterile aqueous solution, or may be administered in a fat-soluble excipient, or in another solution, suspension, patch, tablet or paste format as is appropriate. A composition comprising the compounds described herein may be formulated for administration by inhalation. For instance, a compound may be combined with an excipient to allow dispersion in an aerosol. Examples of inhalation formulations will be known to those skilled in the art. Other agents may be included in combination with the compounds described herein to aid uptake or metabolism, or delay dispersion within the host, such as in a controlled-release formulation. Examples of controlled release formulations will be known to those of skill in the art, and may include microencapsulation, embolism within a carbohydrate or polymer matrix, and the like. Other methods known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences", (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa.

[0053] The dosage of the compositions or compounds of some embodiments described herein may vary depending on the route of administration (oral, intravenous, inhalation, or the like) and the form in which the composition or compound is administered (solution, controlled release or the like). Determination of appropriate dosages is within the ability of one of skill in the art. As used herein, an "effective amount", a "therapeutically effective amount", or a "pharmacologically effective amount" of a compound refers to an amount of the dASO present in such a concentration to result in a therapeutic level of the compound delivered over the term that the compound is used. This may be dependent on the mode of delivery, time period of the dosage, age, weight, general health, sex and diet of the subject receiving the compound. Methods of determining effective amounts are known in the art. It is understood that it could be potentially beneficial to restrict delivery of the compounds described herein to the target tissue or cell in which inhibition of BIRC6 expression and/or the inhibition of cIAPi expression is desired. It is also understood that it may be desirable to target the compounds described herein to a desired tissue or cell type. The compounds described herein may thus be coupled to a targeting moiety. The compounds may be coupled to a cell uptake moiety. The targeting moiety may also function as the cell uptake moiety.

[0054] In general, antisense oligonucleotides as described herein may be used without causing substantial toxicity. Toxicity of the compounds as described herein can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be appropriate to administer substantial excesses of the compositions. Some antisense oligonucleotides as described herein may be toxic at some concentrations. Titration studies may be used to determine toxic and non toxic concentrations. Toxicity may be evaluated by examining a particular antisense oligonucleotide's specificity across cell lines. Animal studies may be used to provide an indication if the compound has any effects on other tissues.

[0055] In some embodiments, antisense oligonucleotides as described herein may be used, for example, and without limitation, in combination with other treatment methods for at least one indication selected from malignancies in which elevated expression of BIRC6 or cIAPi is observed. These include, but are not limited to prostate cancer, childhood de novo acute myeloid leukemia, colorectal cancer, neuroblastoma, melanoma, liver cancer, non-small cell lung cancer, epithelial ovarian cancer, hepatocellular carcinoma, esophageal squamous cell carcinoma, drug- resistant chronic myelogenous leukemia (CML) and breast cancer in mammals, including humans. For example, antisense oligonucleotides and may be used as neoadjuvant (prior), adjunctive (during), and/or adjuvant (after) therapy with surgery, radiation (brachytherapy or external beam), or other therapies (eg. HIFU).

METHODS AND MATERIALS

[0056] The following methods and materials were employed with respect to the EXAMPLES described herein.

[0057] Cell lines

[0058] PC-3 human prostate cancer cell lines were obtained from the American Type Culture Collection (1991, ATCC). C4-2 cells were kindly provided by Dr. L.W.K. Chung (1992, MD Anderson Cancer Center, Houston, Tx). They were maintained as monolayer cultures in RPMI- 1640 (Gibco BRL™, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS). Prior to usage, cells were determined to be mycoplasma free (Mycoplasma Detection Kit™,

Invitrogen™ # rep-pt2) and were not authenticated.

[0059] Tissue microarray (TMA) construction and immunohisto chemistry (IHC)

[0060] Prostate specimens (60 benign prostate samples, 137 primary tumors with no lymph node metastasis, 30 primary tumors with lymph node metastasis, 65 neo-adjuvant treated primary tumors, 67 CRPCs) were obtained from the Vancouver Prostate Centre Tissue Bank following written informed patients' consent and institutional study approval. All samples had been obtained through radical prostatectomy except the CRPC samples that were obtained through transurethral resection of prostate (TURP). TMAs were constructed as previously described (Thomas C, et al, Molecular cancer therapeutics. 2011; 10(2)1347-359).

Immunohistochemical staining using rabbit polyclonal antibody against BIRC6, monoclonal antibody against cIAPi and rabbit polyclonal antibody against XIAP was conducted using a Ventana autostainer (model Discover XT™; Ventana Medical Systems™, Tucson, AZ) with an enzyme-labelled biotin-streptavidin system and a solvent-resistant DAB Map kit (Ventana™). Descriptively, o represents no staining by any tumor cells, 1 represents a faint or focal, questionably present stain, 2 represents a stain of convincing intensity in a minority of cells and 3 a stain of convincing intensity in a majority of cells. [0061 ] Dual IAP-targeting ASO design and validation

[0062] Dual IAP-targeting ASOs (dASOs) were designed in various lengths from 14 to 21 nucleotides with perfect complementary matches to BIRC6 mRNA sections and containing no more than 3 base mismatches to the second target mRNA (i.e. cIAPi). Sequence alignment to each pair of targeted genes was performed using Clustalw

(http://www.genome.jp/tools/clustalw/) and BLAST 2 Sequence in NCBI

(http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) to identify sequences with highest complementarities. ASOs with full phosphorothioate-modified backbone were designed. The dASO knock-down efficacy of the designed dASOs was tested by determining target protein expression 48 hours after transfection using Western blot analysis.

[0063] A person of skill in the art based on the general knowledge in the art and the information provided herein would be able to synthesize the dual -targeting ASOs described herein or modify the dASOs described herein.

[0064] siRNA and ASO transfections

[0065] Small interfering RNAs (siRNAs) targeting cIAPi (si-cIAPi, siGENOME

SMARTpool™ human BIRC2), BIRC6 [si-BIRC6, 5'-GUU-UCA-AAG-CAG-GAU-GAU-G-dTdT- 3'] (Ren J, et ah, Proc Natl Acad Sci U S A. 2005; i02(3):505-570) and negative control (siCtrl) siRNAs were purchased from Dharmacon™ (Cat #M-004390-02-0005, M-003459-03-0005 and D001810-10-05, Chicago, IL). Cells were transfected with siRNA (si-cIAPi, 10 nM for si- BIRC6) or ASO (100-200 nM) for 72 hours using oligofectamin reagent (Invitrogen™) following the manufacturer's instructions.

[0066] Western blotting

[0067] Cell lysates were prepared using cell lysis buffer (1% NP-40, 0.5% sodium deoxycholic acid) supplemented with a protease inhibitor cocktail (Roche™, Nutley, NJ). For detection of BIRC6 (528 kDa), 10 μg whole cell lysate was resolved in 5% SDS-polyacrylamide gel and electrotransferred to a PVDF membrane in tris (25 mM), glycine (191.5 mM), methanol (10%), SDS (0.05%) buffer at 40V overnight at 4°C. Membranes were probed with anti-BIRC6 antibody at room temperature for 2.5 hours. For detection of cIAPi, lysate was resolved in 10% and 15% SDS-polyacrylamide gel, respectively, and electro-transferred to a PVDF membrane in tris (25 mM), glycine (191.5 mM), methanol (10%) buffer at 100V for 1 hour. Membranes were probed with anti-cIAPi antibodies at room temperature for 2.5 hours. Actin or vinculin were used as loading controls and detected on membranes using rabbit anti-actin polyclonal antibody or mouse anti-vinculin antibody.

[0068] Annexin V assay

[0069] Apoptosis was detected by fluorescence-activated cell sorter (FACS) analysis with annexin-V conjugated with fluorescein isothiocyanate (Annexin-V-FITC) (Invitrogen™) and propidium iodide (PI) staining following the manufacturer's protocol as previously described (Low CG et ah, PLoS One. 2013; 8(2)1655837). Early apoptotic cells were identified as Annexin- V positive, PI negative. Data are presented as means ± SD of triplicate experiments.

[0070] MTS cell viability assay

[0071] C4-2 cells (1 x 105) or PC-3 cells (2.5 x 104) were seeded onto 12- well or 24-well culture plates and transfected the next day. MTS (Promega™, Madison, MI) was added to wells at o, 48, 72 and 96 hours after transfection and incubated for 2 hours at 37°C. Aliquots (100 μΐ) of the culture medium were transferred to a 96-well plate for measuring absorbance at OD490. Triplicate wells were tested per assay and each experiment was repeated twice.

[0072] Cell proliferation assay

[0073] PC-3 cells (5 x 104) were seeded onto 12-well plates and transfected with ASOs the next day. Cell numbers were counted at o, 48, 72, 96 hours after transfection using a TC10™ Automated Cell Counter (Bio-rad Laboratories™, Inc, Berkeley, CA). Triplicate wells were tested per assay and the experiment was repeated twice. Results are presented as percentage of untreated control values, mean ± S.D.

[0074] Cell cycle analysis

[0075] Cell cycle distribution was determined by flow cytometry of Pi-stained cells as previously described (Low CG et ah, PLoS One. 2013; 8(2)1655837). Cells were fixed at 72 hours after transfection. The proportion of cells in Gi, S, and G2-M phases of the cell cycle was determined using a FlowJo program™ (TreeStar Inc™, Ashland, OR).

[0076] 4,6-Diamidino-2-phenylindole (DAPI) staining

[0077] PC-3 cells were seeded on cover slips in 12 well-plates and transfected with ASO the next day. After 72 hours of transfection, cells were washed twice with PBS and slides were mounted using VECTASHIELD™ Mounting Medium with DAPI™ (Vector Laboratories™, CA). Cell morphology was examined under a fluorescent microscope (Carl Zeiss™, Germany). Cells exhibiting fragmented nuclear bodies were considered to be undergoing apoptosis. A total of 500 cells were counted in five randomly selected fields per sample using a magnification of 400X.

[0078] Dual luciferase reporter assay

[0079] PC-3 cells (7x103) were seeded onto 96-well plates and co-transfected the next day with 0.05 μg PGL4.32 [luc2P/NF-kB-RE/Hygro] (# E849A, Promega Corp.™, Madison, WI), 1 ng pRL-CMV (Renilla™) and 100 nM dASOs or 10 nM si-BIRC6 or 2 nM si-cIAPi using lipofectamine 2000, following the manufacturer's instructions. Cells were incubated with 20 ng/ ml TNFa for 5 hours at 37°C for induction of NFKB signalling. Luciferase activity was assessed with a Dual-luciferase reporter assay system (#Ei9io, Promega™) at 48 hours after transfection and measured using a Tecan™, Infinite 20oPro™ microplate reader (Tecan™, Mannedorf, Switzerland) following the manufacturer's instructions. Transfection efficiency was normalized to Renilla luciferase activity. Fold induction of NFKB signaling was calculated as average normalized relative light units of induced cells/average normalized relative light units of non-induced cells. Triplicate wells were tested per assay and the experiment was done in duplicate.

[0080] Animal studies

[0081] PC-3 cells (1 x 106) were mixed with matrigel and inoculated subcutaneously in both flanks of 6- to 8-weeks-old NOD-SCID mice under isoflurane anesthesia. When tumors reached a volume of 50-70 mm3, mice were randomized into 3 groups (n = 12 tumors per group), control ASO, dASOs derived from 6w2. The ASOs were administrated to the mice by intraperitoneal injection once daily for 15 consecutive days at a dose of 10 mg/kg. Tumor volume was measured on day o and on day 15, the last day of treatment, using the formula: volume = (width)2 x length/2. Mice were euthanized on day 15 and tumors fixed for immunohistochemical staining. Percentage of tumor growth represents the change in tumor volume measured on days 1 and 15. Viable tumor volume refers to total tumor volume x (100% - % of necrotic area), where % of necrotic area was determined by microscopic examination of H&E stained sections. Scoring of BIRC6 was determined on a four-point scale as mentioned above. Ki-67 positive cells were counted in 6-8 randomly selected fields (40 x magnification) and results are presented as percentage of cells with Ki-67 positive nuclei compared to the total number of cells. Statistical analyses Comparisons of two groups were made using the Student t test. Analyses of correlation between IAP members were performed using a Spearman non-parametric test. Analyses of correlation between BIRC6 expression trend and various prognostic factors were carried out using the Chi square test for trend. Statistical analyses were performed using GraphPad Prism™ 4.0 (GraphPad™). Results with a p<0.05 were considered significant.

[0082] Patient Derived Xenograft (PDX) Studies

[0083] The responses of a panel of 8 PDX models to ENZ were examined. One of the models, LTL313BR, was a castration relapsed subline of patient derived prostate cancer cells which was found to be highly resistant to ENZ. Development of PDX LTL 313B and LTL313BR tumor lines. LTL313BR was accomplished using castration relapse (3-4 months post castration) of parental LTL313B (www.livingtumorlab.com).

[0084] Its parental hormone naive tumor line LTL313B was sensitive to ENZ. The dASO 6w2 was tested for its ability to impede ENZ-resistant CRPC growth. The expression of BIRC6 in relation to ENZ resistance (LTL313B vs LTL313BR) was examined, followed by efficacy study using a larger cohort (N=3o) of ENZ-resistant LTL313BR. Finally, the effect of dASO treatment on LTL313BR apoptosis and survival signaling were also investigated.

EXAMPLES

[0085] EXAMPLE 1: ASO-6w2 length variants (Gen 1.0) transfection showed varying BIRC6 target inhibition ability

[0086] The target mRNA inhibition ability and specificity of ASO-6w2 and its length variants were assessed using first generation ASO. 15 ASOs length variants (from 14-21 nucleotides) in closest proximity to original ASO-6w2 (19 nucleotides) were transfected into PC-3 cells (see TABLE A). Cells were collected 48 hours after transfection and BIRC6 mRNA level was examined by qPCR. As shown in FIGURE 1, ASO-6w2 length variants transfected cells exhibited varying level of BIRC6 expressions. Majority of variants did not resulted in significant BIRC6 inhibition except for the original ASO-6w2 (i9nt) and i8nt V5. Surprisingly, a few transfected variants resulted in increased BIRC6 levels, such as i8nt v4, I7nt v6 and I5nt v2. The results suggest that ASO targeting activity is difficult to predict based on sequence homology.

[0087] EXAMPLE 2: Generation 2.5 Dual Antisense Oligonucleotides (dASO)- i6mer 6w2 Targeting BIRC6 and cIAPi in vitro and in vivo

[0088] Based on the earlier efficacy data of the first generation (phosphorothioate backbone) dASO-6w2, new generation dASOs were synthesized for improved clinical usage (Ionis Pharm.™, Carlsbad, CA) and tested for anti-tumour efficacy. The latest generation dASOs (Gen2.5) are chemically modified to contain 2'-4' constrained ethyl (cET) modified bicyclic nucleic acid oligonucleotides flanking a central DNA gap (Donner, Yeh et al. 2015; Yamamoto, Loriot et al. 2015). The Gen2.5 dASOs-6w2 is 16-nt long, 3-nt shorter than the first generation dASO (19-nt). They have significantly enhanced stability in vivo and potency compared to the previous dASO generation and can therefore be considered for clinical use without further formulation (Revenko, Ross et al. 2015).

[0089] Four Gen2.5 dASO-6w2 variants were synthesized (for sequences, see TABLE A— SEQ ID NOs:i-4) and first tested for target inhibition in vitro. Gen2.5 dASOs-6w2 (A-D) were transfected into human PC-3 prostate cancer cells and the expressions of BIRC6 and cIAPi at gene and protein levels examined. As shown in FIGURES 2A, 2B, 3A and 3B, all four Gen2.5 dASO variants markedly inhibited the expressions of BIRC6 and cIAPi genes and proteins. They were also shown to markedly suppress the proliferation of PC-3 cells in vitro (FIGURE 4). As the four drug candidates were quite similar in efficacy, Gen 2.5 dASO-6w2-A (SEQ ID NO:i) was selected for further in vivo testing based on its ability to silence both BIRC6 & cIAPi genes in a dose- dependent manner, suggesting a superior target specificity.

[0090] EXAMPLE 3: Gen2.5 dASO-6w2-A Markedly Suppresses Growth of Multiple Clinically Relevant Prostate Cancer PDX Models

[0091 ] To evaluate the therapeutic potential of Gen2.5 dASO-6w2-A in vivo, we used patient- derived xenograft (PDX) models of prostate cancer tissues as it has recently been recognized that such cancer tissue-based xenograft models have more accurate predictive ability, and hence clinical relevance, than commonly used cell line-based models. Thus, in contrast to the latter, they retain histopathological properties of the original cancers, such as tumour heterogeneity and tumour-stroma architecture, important factors in cancer growth and progression. At the Living Tumour Laboratory (LTL) of the BC Cancer Agency, we have established 'high fidelity' transplantable prostate tumour xenograft lines by grafting, and transplanting, patients' tumour tissue in NOD-SCID mice at the renal graft site (Choi, Lin et al. 2014; Lin, Wyatt et al. 2014). The LTL prostate cancer repository encompasses models of primary prostate adenocarcinoma, castration-resistant prostate cancer (CRPC), enzalutamide-resistant CRPC and neuroendocrine prostate cancer (NEPC) (www.livingtumourlab.ca). These stable LTL PDX tumour tissue lines represent an excellent platform for drug efficacy testing, particularly of advanced, currently incurable prostate cancer subtypes. The anticancer activity of Gen2.5 dASO-6w2-A was tested using PDX models of the above four prostate cancer subtypes in parallel. Several research groups have reported use of multiple PDX models of various cancer types for testing drug efficacy (Migliardi, Sassi et al. 2012; Gao, Korn et al. 2015; Krepler, Xiao et al. 2016).

[0092] A pilot 'preclinical PDX trial' was carried out with Gen2.5 dASO-6w2-A, using the following eight PDX lines of prostate cancer subtypes: LTL-471, LTL-484, LTL-412, LTL-555 and LTL-573 (hormone naive adenocarcinoma); LTL-573R (CRPC), LTL-313HR (enzalutamide- resistant CRPC) and LTL-610 (NEPC). Each line includes minimal of 1 mouse in a Gen2.5 dASO- 6w2-A treatment group and minimal of 1 mouse in the control ASO treatment group. Control (Ctrl) ASO has the same chemistry as Gen2.5 dASO-6w2-A, sequence: GGCTACTACGCCGTCA (provided by Ionis Pharm.™). All PDX tumours were grafted subcutaneously (s.c), except for LTL-555 and LTL-412 tumours which were grafted under the subrenal capsule (src). When tumours reached a volume of about 100 mm3, mice were treated with Gen2.5 dASO-6w2-A/Ctrl ASO, with a loading dose of 30 mg/kg on the first day, followed by a 15 mg/kg daily maintenance dose for the following 20 days in LTL-573R, LTL-610, LTL-573, LTL-313HR (13 days for LTL- 555)- loading dose of 30 mg/kg on the first day, followed by 15 mg/kg maintenance dose 5 times a week for 3 weeks were administrated in LTL-471, LTL-484, LTL-412 (See TABLE B). Tumours in the sc site were measured by caliper twice a week, until 1-2 weeks after the end of the treatment. Blood plasma was collected from LTL-555 and LTL-412 tumour-bearing mice (src grafted models) once a week for monitoring PSA levels as an indication of tumour growth. Mice were euthanized and tumours were harvested at 1-2 weeks after the end of treatment. The tumour growth rates were calculated from the slopes of the log-scale tumour growth plots (not shown).

[0093] As shown in FIGURES 5 and 6, treatment with Gen2.5 dASO-6w2-A led to tumour growth inhibition in multiple PDX models tested, with most promising suppressive effects in LTL- 471 (hormone naive PCa), LTL-573R (CRPC) and LTL-313HR (enzalutamide-resistant CRPC) models (T/C growth rate ratios, 0.23, 0.26 and 0.27, respectively). Mild suppressions were observed in the dASO-6w2-A-treated LTL-555 (hormone naive PCa), LTL-484 (hormone naive PCa) and LTL-610 (NEPC) models, with T/C of 0.45, 0.62 and 0.73, respectively (FIGURE 6).

[0094] TABLE B. The T/C ratios, growth rates and treatment regimens detail of PDX lines. In SC grafted lines, growth rates(u)were calculated from the slopes of the tumour size (log 10) growth plots during the treatment period (not shown). T/C ratio of tumour volume were calculated by the equation io^A(uT-uC)x measurement days. In SRC grafted lines (LTL-555 and LTL-412), T/ C ratio of tumour volume was calculated based on the tumor volumes at harvest. SC, subcutaneous; SRC, subrenal capsule. Treatment measurement μ (growth

PDX line site regimen days group n rate) T/C

1 Cont' 21 Control 1 0.0695 0.262

573R SC CRPC 15

days 6w2-A 1 0.0307

2 Cont' 21 Control 2 0.0325 0.730

610 SC NEPC 24

days 6w2-A 2 0.0268

3 Cont' 21 Control 1 0.0136 0.914

573 SC PCa 26

days 6w2-A 1 0.0121

4 Cont' 21 Control 3(2mice) 0.0187 0.268

313HR SC CRPC 29

days 6w2-A 3(2mice) -0.0010

5 5 QD per wk Control 1 0.0225 0.233

471 SC PCa 29

x3 6w2-A 1 0.0007

6 5 QD per wk Control 1 0.0260 0.623

484 SC PCa 15

x3 6w2-A 1 0.0123

Treatment mean vol.

PDX line site regimen Harvest day group n (at harves it) T/C

7 Cont' 15 Control 8 (2mice) 318.37 0.454

555 SRC PCa days, rest 1 21

6w2-A 8 (2mice) 144.51

week

8 5 QD per wk Control 2 (2mice) 385.24 0.591

412 SRC PCa 29

x3 6w2-A 2 (2mice) 227.64

[0095] No major weight loss was associated with the treatment with Gen2.5 dASO-6w2-A of the LTL-573R and LTL-610 models (FIGURE 7). The LTL-555 (subrenal capsule model) and LTL-573 models showed about 20% reduction in body weight in Gen2.5 dASO-6w2-A-treated mice.

[0096] EXAMPLE 4: Increased Apoptosis and Defective Vasculature in Tumours of Gen2.5 dASO-6w2-A-Treated PDX Models

[0097] Gen2.5 dASO-6w2-A-treated tumours showed increased apoptosis in the LTL-610 and LTL-573R lines and slightly in the LTL-555 line, as indicated by cleaved caspase-3 immunohistochemistry (IHC) staining (FIGURE 8). The increase in tumour apoptosis in the treated mice is in consistent with previous results in PC-3 and LTL-313BR in vivo models using Gen 1 6w2 ASO. The increased tumour cell death following treatment with Gen2.5 dASO-6w2-A could contribute, at least in part, to the reduction of PDX tumour growth. The result suggests that BIRC6-cIAPi-targeting ASOs could be effective against various types of prostate cancer (hormone naive prostate cancer, CRPC and NEPC), irrespective of their degree of androgen dependency.

[0098] Moreover, tumour H& E staining revealed a distinguished pattern of defective vasculature in the treated tumours (FIGURE 9). The haemorrhage, as a result of leaking vasculature, resembles that of the 'tomato phenotype' identified in cIAPi null mutant zebrafish embryos (Santoro, Samuel et al. 2007; Lopez, John et al. 2011). The phenomenon was consistently seen in various PDX models treated with Gen2.5 dASO-6w2-A but not in the Ctrl ASO group. The defective vasculature in tumours of Gen2.5 dASO-6w2-A-treated mice may contribute to the reduced growth and viability of the tumours (due to lower actual tumour cell content).

[0099] EXAMPLE 5: Gen2.5 6w2-A treatment effectively inhibits tumour BIRC6 mRNA expression

[00100] The expressions of target genes in tumours of control and Gen2.5 dASO-6w2-A- treated mice immediately at the end of the treatment in were examined in two representative PDX lines. As shown in FIGURE 10, qPCR analysis showed that Gen2.5 6w2-A inhibits tumour BIRC6 and cIAPi mRNA in LTL-573R (Ctrl n = 2, 6w2-A n= 3) and BIRC6 in LTL-484 (Ctrl n = 1, 6w2-A n= 1).

[00101] In summary, we have provided evidence that a BIRC6-cIAPl dual targeting ASO, as single agent, is capable of suppressing the growth of primary and advanced prostate cancer, i.e. hormone naive prostate cancer, CRPC, ENZ -resistant CRPC, as well as NEPC. It is expected that a combination of the dASO with other therapies such as chemotherapies, androgen deprivation therapies, second- generation anti-androgens (e.g., enzalutamide, etc.) can enhance the therapeutic efficacy, and potentially improve survival of patients with various forms of prostate cancer.

[00102] Although embodiments described herein have been described in some detail by way of illustration and example for the purposes of clarity of understanding, it will be readily apparent to those of skill in the art in light of the teachings described herein that changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word "comprising" is used herein as an open ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as herein described and with reference to the figures. [00103] REFERENCES

[00104] Bartke T, Pohl C, Pyrowolakis G and Jentsch S. Dual role of BRUCE as an antiapoptotic IAP and a chimeric E2/E3 ubiquitin ligase. Mol Cell. 2004; i4(6):8oi-8n.

[00105] Bianchini M, Levy E, Zucchini C, Pinski V, Macagno C, De Sanctis P, Valvassori L,

Carinci P and Mordoh J. Comparative study of gene expression by cDNA microarray in human colorectal cancer tissues and normal mucosa. Int J Oncol. 2006; 29(i):83-94.

[00106] Bishr M and Saad F. Overview of the latest treatments for castration-resistant prostate cancer. Nat Rev Urol. 2013; io(9):522-528.

[00107] Chen Z, Naito M, Hori S, Mashima T, Yamori T and Tsuruo T. A human IAP-f amily gene, apollon, expressed in human brain cancer cells. Biochem Biophys Res Commun. 1999; 264(3):847-854·

[00108] Cheung HH, Plenchette S, Kern CJ, Mahoney DJ and Korneluk RG. The RING domain of cIAPi mediates the degradation of RING-bearing inhibitor of apoptosis proteins by distinct pathways. Mol Biol Cell. 2008; i9(7):2729-2740.

[00109] Choi, S. Y., D. Lin, et al. (2014). "Lessons from patient-derived xenografts for better in vitro modeling of human cancer." Adv Drug Deliv Rev 79-80: 222-237.

[00110] Chu L, Gu J, Sun L, Qian Q, Qian C and Liu X. Oncolytic adenovirus-mediated shRNA against Apollon inhibits tumor cell growth and enhances antitumor effect of 5- fluorouracil. Gene Ther. 2008; i5(7):484-494.

[00111] Claessens F, Helsen C, Prekovic S, Van den Broeck T, Spans L, Van Poppel H and Joniau S. Emerging mechanisms of enzalutamide resistance in prostate cancer. Nat Rev Urol. 2014; Ii(i2):7i2-7i6.

[00112] Dai Y, Liu M, Tang W, DeSano J, Burstein E, Davis M, Pienta K, Lawrence T and Xu L. Molecularly targeted radiosensitization of human prostate cancer by modulating inhibitor of apoptosis. Clin Cancer Res. 2008; i4(23):770i-77io.

[00113] de Almagro MC and Vucic D. The inhibitor of apoptosis (IAP) proteins are critical regulators of signaling pathways and targets for anti-cancer therapy. Exp Oncol. 2012; 34(3)1200- 211.

[00114] Dias N and Stein CA. Antisense oligonucleotides: basic concepts and mechanisms. Mol Cancer Ther. 2002; ι(5):347-355·

[00115] Dong X, Lin D, Low C, Vucic EA, English JC, Yee J, Murray N, Lam WL, Ling V, Lam S, Gout PW and Wang Y. Elevated expression of BIRC6 protein in non-small-cell lung cancers is associated with cancer recurrence and chemoresistance. J Thorac Oncol. 2013; 8(2): 161-170. [00116] Donner, A. J., S. T. Yeh, et al. (2015). "CD40 Generation 2.5 Antisense Oligonucleotide Treatment Attenuates Doxorubicin-induced Nephropathy and Kidney Inflammation." Mol Ther Nucleic Acids 4: e205.

[00117] Gao, H., J. M. Korn, et al. (2015). "High-throughput screening using patient- derived tumor xenografts to predict clinical trial drug response." Nat Med 21(11): 1318-1325.

[00118] Gleave M, Miyake H and Chi K. Beyond simple castration: targeting the molecular basis of treatment resistance in advanced prostate cancer. Cancer Chemother Pharmacol. 2005; 56 Suppl ΐ:47-57·

[00119] Gyrd-Hansen M and Meier P. IAPs: from caspase inhibitors to modulators of NF- kappaB, inflammation and cancer. Nat Rev Cancer. 2010; io(8):56i-574.

[00120] Hao Y, Sekine K, Kawabata A, Nakamura H, Ishioka T, Ohata H, Katayama R,

Hashimoto C, Zhang X, Noda T, Tsuruo T and Naito M. Apollon ubiquitinates SMAC and caspase-

9, and has an essential cytoprotection function. Nat Cell Biol. 2004; 6(9):849-86o.

[00121] HensleyP, Mishra M and Kyprianou N. Targeting caspases in cancer therapeutics.

Biol Chem. 2013; 394(7):83i-843.

[00122] Krajewska M, Krajewski S, Banares S, Huang X, Turner B, Bubendorf L, Kallioniemi OP, Shabaik A, Vitiello A, Peehl D, Gao GJ and Reed JC. Elevated expression of inhibitor of apoptosis proteins in prostate cancer. Clin Cancer Res. 2003; 9(i3):49i4-4925.

[00123] Krepler, C, M. Xiao, et al. (2016). "Personalized Preclinical Trials in BRAF Inhibitor-Resistant Patient-Derived Xenograft Models Identify Second-Line Combination Therapies." Clin Cancer Res 22(7): 1592-1602.

[00124] Lamers F, Schild L, Koster J, Speleman F, Ora I, Westerhout EM, van Sluis P, Versteeg R, Caron HN and Molenaar JJ. Identification of BIRC6 as a novel intervention target for neuroblastoma therapy. BMC Cancer. 2012; 12:285.

[00125] Lin D, Gout PW and Wang Y. Lessons from in-vivo models of castration-resistant prostate cancer. Curr Opin Urol. 2013; 23(3):214-219.

[00126] Lin D, Wyatt AW, Xue H, Wang Y, Dong X, Haegert A, Wu R, Brahmbhatt S, Mo F, Jong L, Bell RH, Anderson S, Hurtado-Coll A, Fazli L, Sharma M, Beltran H, et al. High fidelity patient-derived xenografts for accelerating prostate cancer discovery and drug development. Cancer Res. 2014; 74(4):i272-i283.

[00127] Lopez, J., S. W. John, et al. (2011). "CARD-mediated autoinhibition of cIAPi's E3 ligase activity suppresses cell proliferation and migration." Mol Cell 42(5): 569-583. [00128] Low CG, Luk IS, Lin D, Fazli L, Yang K, Xu Y, Gleave M, Gout PW and Wang Y. BIRC6 protein, an inhibitor of apoptosis: role in survival of human prostate cancer cells. PLoS One. 2013; 8(2):e55837.

[00129] Martin SJ. An Apollon vista of death and destruction. Nat Cell Biol. 2004; 6(9):8o4-8o6.

[00130] Micheau, O. and J. Tschopp (2003). "Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes." Cell 114(2): 181-190.

[00131] Migliardi, G., F. Sassi, et al. (2012). "Inhibition of MEK and Pl3K/mTOR suppresses tumor growth but does not cause tumor regression in patient-derived xenografts of PvAS-mutant colorectal carcinomas." Clin Cancer Res 18(9): 2515-2525.

[00132] Pan WH, Clawson GA. Identifying accessible sites in RNA: the first step in designing antisense reagents. Curr Med Chem. 20o6;i3(25):3o83-i03.

[00133] Patzel V. In silico selection of active siRNA. Drug Discov Today. 2007 Feb;i2(3- 4):i39-48.

[00134] Peek AS, Behlke MA. Design of active small interfering RNAs. Curr Opin Mol Ther. 2007 Apr;9(2):iio-8.

[00135] Petrylak DP, Tangen CM, Hussain MH, Lara PN, Jr., Jones JA, Taplin ME, Burch PA, Berry D, Moinpour C, Kohli M, Benson MC, Small EJ, Raghavan D and Crawford ED. Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. N Engl J Med. 2004; 35i(i5):i5i3-i520.

[00136] Pohl C and Jentsch S. Final stages of cytokinesis and midbody ring formation are controlled by BRUCE. Cell. 2008; i32(s):832-845.

[00137] Qiu XB, Markant SL, Yuan J and Goldberg AL. Nrdpi-mediated degradation of the gigantic IAP, BRUCE, is a novel pathway for triggering apoptosis. EMBO J. 2004; 23(4):8oo-8io.

[00138] Qiu XB and Goldberg AL. The membrane-associated inhibitor of apoptosis protein, BRUCE/Apollon, antagonizes both the precursor and mature forms of Smac and caspase-9. J Biol Chem. 2005; 280(1): 174-182.

[00139] Revenko, A. S., S. J. Ross, et al. (2015). "Abstract PR12: Discovery and preclinical evaluation of cEt-modified KRAS antisense oligonucleotide inhibitors." American Association for Cancer Research 14(12 Supplement 2): PR12-PR12.

[00140] Ren J, Shi M, Liu R, Yang QH, Johnson T, Skarnes WC and Du C. The Birc6 (Bruce) gene regulates P53 and the mitochondrial pathway of apoptosis and is essential for mouse embryonic development. Proc Natl Acad Sci U S A. 2005; i02(3):505-570. [00141] Santoro, M. M., T. Samuel, et al. (2007). "Birc2 (clapi) regulates endothelial cell integrity and blood vessel homeostasis." Nat Genet 39(11): 1397-1402.

[00142] Sekine K, Hao Y, Suzuki Y, Takahashi R, Tsuruo T and Naito M. HtrA2 cleaves Apollon and induces cell death by LAP-binding motif in Apollon-deficient cells. Biochem Biophys Res Commun. 2005; 33θ(ι):279-285.

[00143] Siegel R, Naishadham D and Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012; 62(i):io-29.

[00144] Sung KW, Choi J, Hwang YK, Lee SJ, Kim HJ, Lee SH, Yoo KH, Jung HL and Koo HH. Overexpression of Apollon, an antiapoptotic protein, is associated with poor prognosis in childhood de novo acute myeloid leukemia. Clin Cancer Res. 2007; I3(i7):5i09-5ii4.

[00145] Tannock IF, de Wit R, Berry WR, Horti J, Pluzanska A, Chi KN, Oudard S, Theodore C, James ND, Turesson I, Rosenthal MA and Eisenberger MA. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med. 2004; 35i(i5):i502-i5i2.

[00146] Tassi E, Zanon M, Vegetti C, Molla A, Bersani I, Perotti V, Pennati M, Zaffaroni N, Milella M, Ferrone S, Carlo-Stella C, Gianni AM, Mortarini R and Anichini A. Role of Apollon in human melanoma resistance to antitumor agents that activate the intrinsic or the extrinsic apoptosis pathways. Clin Cancer Res. 2012; i8(i2):33i6-3327.

[00147] Thomas C, Zoubeidi A, Kuruma H, Fazli L, Lamoureux F, Beraldi E, Monia BP, MacLeod AR, Thuroff JW and Gleave ME. Transcription factor Stat5 knockdown enhances androgen receptor degradation and delays castration-resistant prostate cancer progression in vivo. Molecular cancer therapeutics. 2011; io(2):347-359.

[00148] Yamamoto, Y., Y. Loriot, et al. (2015). "Generation 2.5 antisense oligonucleotides targeting the androgen receptor and its splice variants suppress enzalutamide-resistant prostate cancer cell growth." Clin Cancer Res 21(7): 1675-1687.

[00149] Zielinski RR, Eigl BJ and Chi KN. Targeting the apoptosis pathway in prostate cancer. Cancer J. 2013; i9(i):79-89. INFORMAL SEQUENCE LISTING SEQ ID NO : 1

Description: dASO 6w2 delta A (16me CAGCATCATGTGGACT

SEQ ID NO: 2

Description: dASO 6w2 delta B (16me GCAGCATCATGTGGAC

SEQ ID NO: 3

Description: dASO 6w2 delta C (16me TGCAGC TCATGTGGA

SEQ ID NO: 4

Description: dASO 6w2 delta D (16me CTGCAGCATCATGTGG

SEQ ID NO: 5

Description: Control dASO (16mer) GGCTACTACGCCGTCA

SEQ ID NO : 6

Description: dASO 6w2 (19mer)

CTGCAGCATCATGTGGACT

SEQ ID NO : 7

Description: dASO 21nt-vl (21mer) CTGCAGCATCATGTGGACTGT

SEQ ID NO: 8

Description: dASO 20nt-v2 (20mer) GCTGCAGCATCATGTGGACT

SEQ ID NO : 9

Description: dASO 20nt-v3 (20mer) CTGCAGCATCATGTGGACTG

SEQ ID NO: 10

Description: dASO 18nt-v4 (18mer) TGCAGCATCATGTGGACT

SEQ ID NO: 11

Description: dASO 18nt-v5 (18mer) CTGCAGCATCATGTGGAC

SEQ ID NO: 12

Description: dASO 17nt-v6 (17mer) GCAGCATCATGTGGACT SEQ ID NO: 13

Description: dASO 17nt-v7 (17mer)

CTGCAGCATCATGTGGA

SEQ ID NO: 14

Description: dASO 17nt-v8 (17mer)

TGCAGCATCATGTGGAC

SEQ ID NO: 15

Description: dASO 15nt-v9 (15mer)

AGCATCATGTGGACT

SEQ ID NO: 16

Description: dASO 15nt-vl0 (15mer)

CAGCATCATGTGGAC

SEQ ID NO: 17

Description: dASO 15nt-vll (15mer)

GCAGCATCATGTGGA

SEQ ID NO: 18

Description: dASO 15nt-vl2 (15mer)

TGCAGCATCATGTGG

SEQ ID NO: 19

Description: dASO 15nt-vl3 (15mer)

CTGCAGCATCATGTG

SEQ ID NO: 20

Description: dASO 14nt-vl (14mer)

TGCAGCATCATGTG

SEQ ID NO: 21

Description: Scramble (Serb) B control (Non-targeting control ASO) 5' CCTTCCCTGAAGGTTCCTCC 3'

Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer, the method comprising administering a dual-targeting

antisense oligonucleotides (dASO) of SEQ ID NOs:i-4, 7-9, 11, 15, 17 or 20.

2. The method of claim 1, dual-targeting antisense oligonucleotides (dASO) of SEQ ID NOs:

1-4, 7, 8, 11 or 15.

3. The method of claim 1 or 2, dual-targeting antisense oligonucleotides (dASO) of SEQ ID NO:i-4 or 11.

4. The method of claim 1, 2 or 3, wherein the dASO further comprises a modified

internucleoside linkage.

5. The method of claim 4, wherein the modified internucleoside linkage is a peptide-nucleic acid linkage, a morpholino linkage, a N3' to P5' phosphoramidate linkage, a

methylphosphonate linkage or a phosphorothioate linkage.

6. The method of claim 1, 2 or 3, wherein the dASO further comprises a modified sugar moiety.

7. The method of claim 6, wherein the modified sugar moiety is: a 2 ' -O-alkyl

oligoribonucleotide; a constrained ethyl (cEt); or a locked nucleic acid (LNA).

8. The method of claim 1, 2 or 3, wherein the dASO has a 2'MOE gapmer modification.

9. The method of claim 1, 2 or 3, wherein the dASO further comprises a modified

nucleobase.

10. The method of claim 9, wherein the modified nucleobase is a 5-methyl pynmidine or a 5- propynyl pyrimidine.

11. The method of any one of claims 1-10, wherein the cell is a human cell.

12. The method of any one of claims 1-11, wherein the cancer is characterized by elevated expression of one of more of BIRC6 or cIAPi.

13. The method of any one of claims 1-12, wherein the cancer is selected from one or more of the following: prostate cancer; childhood de novo acute myeloid leukemia; colorectal cancer; neuroblastoma; melanoma; liver cancer; non-small cell lung cancer; epithelial ovarian cancer; hepatocellular carcinoma; esophageal squamous cell carcinoma; drug- resistant chronic myelogenous leukemia (CML); and breast cancer.

14. The method of claim 13, wherein the prostate cancer is castration-resistant prostate cancer (CRPC).

15. The method of claim 14, wherein the prostate cancer is enzalutamide (ENZ) resistant CRPC.

16. The method of any one of claims 1-10, wherein the dASO is substantially complementary to the mRNA of BIRC6 and cIAPi.

17. The method of any one of claims 1-16, wherein the dASO is administered intranvenously.

18. The method of any one of claims 1-16, wherein the dASO is topically administered to a tissue.

19. The method of any one of claims 1-16, wherein the dASO is mixed with lipid particles prior to administration.

20. The method of any one of claims 1-16, wherein the dASO is encapsulated in liposomes prior to administration.

21. A dual-targeting antisense oligonucleotide (dASO), wherein the oligonucleotide has a sequence selected from one or more of the following:

(a) CAGCATCATGTGGACT (SEQ ID NO: 1);

(b) GC AGC AT CAT GT GG AC (SEQ ID NO: 2);

(c) TGCAGCATCATGTGGA (SEQ ID NO: 3);

(d) c T GC AGC AT CAT GT GG (SEQ ID NO: 4); and

(e) CTGCAGCATCATGTGGAC (SEQ ID NO: ll).

22. The dASO of claim 21, wherein the dASO further comprises a modified internucleoside linkage.

23. The dASO of claim 22, wherein the modified internucleoside linkage is a peptide-nucleic acid linkage, a morpholino linkage, a N3' to P5' phosphoramidate linkage, a

methylphosphonate linkage or a phosphorothioate linkage.

24. The dASO of claim 21, wherein the dASO further comprises a modified sugar moiety.

25. The dASO of claim 24, wherein the modified sugar moiety is: a 2 ' -O-alkyl

oligoribonucleotide; a constrained ethyl (cEt); or a locked nucleic acid (LNA).

26. The dASO of claim 21, wherein the dASO has a 2'MOE gapmer modification.

27. The dASO of claim 21, wherein the dASO further comprises a modified nucleobase.

28. The dASO of claim 27, wherein the modified nucleobase is a 5-methyl pyrimidine or a 5- propynyl pyrimidine.

29. A pharmaceutical composition, the composition comprising (a) dual-targeting antisense oligonucleotide (dASO) of SEQ ID NO:i, SEQ ID N0:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO: 11; and (b) a pharmaceutically acceptable carrier.

30. The pharmaceutical composition of claim 29, wherein the dASO further comprises a modified internucleoside linkage.

31. The pharmaceutical composition of claim 30, wherein the modified internucleoside linkage is a peptide-nucleic acid linkage, a morpholino linkage, a N3' to P5'

phosphoramidate linkage, a methylphosphonate linkage or a phosphorothioate linkage.

32. The pharmaceutical composition of claim 29, wherein the dASO further comprises a modified sugar moiety.

33. The pharmaceutical composition of claim 31, wherein the dASO further comprises a modified sugar moiety.

34. The pharmaceutical composition of claim 32 or 33, wherein the modified sugar moiety is: a 2 ' -O-alkyl oligoribonucleotide; a constrained ethyl (cEt); or a locked nucleic acid (LNA).

35. The pharmaceutical composition of claim 29, wherein the dASO has a 2'MOE gapmer modification.

36. The pharmaceutical composition of claim 29, wherein the dASO further comprises a modified nucleobase.

37. The pharmaceutical composition of claim 36, wherein the modified nucleobase is a 5- methyl pyrimidine or a 5- propynyl pyrimidine.

38. A commercial package, comprising:

a. a dASO of SEQ ID NO:i, SEQ ID N0:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO: 11; and

b. instructions for the treatment of cancer.

39. The commercial package of claim 38, wherein the cancer is selected from one or more of the following: prostate cancer; childhood de novo acute myeloid leukemia; colorectal cancer; neuroblastoma; melanoma; liver cancer; non-small cell lung cancer; epithelial ovarian cancer; hepatocellular carcinoma; esophageal squamous cell carcinoma; drug- resistant chronic myelogenous leukemia (CML); and breast cancer.

40. The commercial package of claim 38, wherein the prostate cancer is castration-resistant prostate cancer (CRPC).