Determining cancer responsiveness to treatment

Abstract

To provide methods for treating a cancer with an anti-cancer agent capable of inhibiting an enzyme that mediates repair of DNA strand breaks such as PARP, or methods for predicting the responsiveness of a cancer to the anti-cancer agent.SOLUTION: Provided herein is a method including a step of determining an expression level of SASH1. Also provided is a method for treating cancer comprising administering a therapeutically effective amount of an agent that increases the expression and / or an activity of SASH1. Further provided herein is a method for identifying an agent that modulates the expression and / or an activity of SASH1 for use in the treatment of cancer.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to cancer. More specifically, the present invention relates to methods for treating cancer (particularly lung cancer and cancers of the reproductive system (such as breast cancer)) and / or determining the responsiveness of cancer to treatment and / or the prognosis of cancer. [Background technology]

[0002] Breast cancer is the second most common cancer worldwide, accounting for 25% of all cancers in women. Thanks to improved management strategies, survival rates have increased over the past few decades. However, the disease remains the second leading cause of cancer-related deaths in women [1]. Current treatment strategies for breast cancer are primarily based on the pathological characteristics of a patient's tumor. Expression of hormone (estrogen (ER) and progesterone) receptors and human epidermal growth factor 2 (HER2) receptors is routinely assessed at diagnosis because these proteins are currently the strongest prognostic and predictive biomarkers for breast cancer. Treatment options are severely limited for tumors negative for these receptors ("triple-negative") [2, 3]. Therefore, there is a continuing need for the development of new treatments as well as prognostic markers for not only breast cancer but also a broader range of cancers.

[0003] Poly(ADP-ribose) polymerase (PARP) inhibitors (e.g., olaparib) were originally designed to treat BRCA-mutated or triple-negative breast cancer (loss of BRCA1 or BRCA2 function is often referred to as "BRCAness"). However, BRCA1 and BRCA2 have not yet become reliable biomarkers for sensitivity to these therapeutic agents. While "BRACA-inhibitory status" and loss of heterozygosity (LOH) remain complementary diagnostics for patient sensitivity to PARP inhibitors, these readouts are not reliable predictors of drug response. As a result, multiple large-scale phase III clinical trials have been required to demonstrate a survival benefit over progression. Nevertheless, numerous clinical trials investigating the efficacy of PARP inhibitors are currently underway for many types of solid tumors, including ovarian, prostate, breast, and lung cancers. Therefore, there remains a continuing need for improved companion / complementary diagnostics for PARP inhibitor-based cancer treatment.

[0004] SAM domain and SH3 domain containing 1 (SASH1) was initially identified as a putative tumor suppressor gene based on the detection of significantly lower mRNA levels in breast, lung, thyroid, and colorectal cancers compared with adjacent normal tissues [4, 5]. Low SASH1 expression was also correlated with invasive tumor growth, metastasis, and poor prognosis in colorectal cancer [6]. As many as 74% of breast cancers show reduced expression of SASH1 transcripts [4]. This observation was later confirmed by immunohistochemical studies that found reduced SASH1 expression in breast cancers compared with adjacent normal tissues [7]. Methylation of the SASH1 promoter (particularly CpG_26.27 and CpG_54.55) correlates with SASH1 repression in breast cancer [7].

[0005] Although the precise function of SASH1 in normal tissues and cancer remains unclear, the protein is known to be localized in the nucleus, and its SAM and SH3 domains may play a signaling, adaptor, and / or molecular scaffolding role [8, 9]. The suggested tumor-suppressor role of SASH1 is consistent with studies demonstrating that its functional inhibition increases cell survival, proliferation, and migration in A549 lung cancer cells [5, 10-12], while its overexpression significantly increases apoptosis [5]. Additionally, ectopic expression of SASH1 has been shown to promote the expression of apoptotic proteins, including caspase-3

[10] . Aside from studies examining its expression, mutational status, and DNA methylation, the role of SASH1 in breast cancer is poorly understood. Summary of the Invention

[0006] The present invention broadly relates to identifying SASH1 expression levels as a predictive and / or prognostic marker for cancer response to treatment with PARP inhibitors. In some aspects, the present invention also broadly relates to treating cancer with agents that induce and / or increase SASH1 expression. In one particular embodiment, the cancer is a cancer of the reproductive system, such as breast cancer.

[0007] In a first aspect, the present invention provides a method for predicting the responsiveness of a cancer to an anticancer drug in a subject, wherein the anticancer drug is an anticancer drug that at least partially inhibits the activity of an enzyme that mediates the repair of DNA strand breaks, and the method includes a step of determining the expression level of SASH1 protein or a nucleic acid encoding the SASH1 protein in one or more cancer cells, cancer tissues, or cancer organs of the subject, and the expression level of the SASH1 protein or the nucleic acid encoding the SASH1 protein indicates a relative increase or decrease in the responsiveness of the cancer to the anticancer drug, or is correlated with a relative increase or decrease in the responsiveness of the cancer to the anticancer drug.

[0008] In particular embodiments, a relative decrease in the level of SASH1 protein or a nucleic acid encoding a SASH1 protein indicates a relative increase in the cancer's response to an anti-cancer drug or is correlated with a relative increase in the cancer's response to an anti-cancer drug, and / or a relative increase in the level of SASH1 protein or a nucleic acid encoding a SASH1 protein indicates a relative decrease in the cancer's response to an anti-cancer drug or is correlated with a relative decrease in the cancer's response to an anti-cancer drug.

[0009] In one embodiment, the method of the above aspect further comprises treating the subject for cancer.

[0010] In a second aspect, the present invention provides a method for treating cancer in a subject, comprising determining the expression level of SASH1 protein or a nucleic acid encoding SASH1 protein in one or more cancer cells, cancer tissues, or cancer organs of the subject, and initiating, continuing, modifying, or discontinuing cancer treatment based on the results.

[0011] Suitably, the cancer treatment comprises administering a therapeutically effective amount of an anti-cancer agent that inhibits the activity of an enzyme that mediates the repair of DNA strand breaks.

[0012] In some embodiments, the cancer treatment comprises administering to the subject a therapeutically effective amount of an agent that inhibits or blocks the expression and / or activity of SASH1.

[0013] In a third aspect, the present invention provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an agent that inhibits or blocks SASH1 expression and / or activity, in combination with an anti-cancer agent that inhibits the activity of an enzyme that mediates the repair of DNA strand breaks.

[0014] For the first, second and third aspects, the enzyme is suitably poly(ADP-ribose) polymerase (PARP).

[0015] In a fourth aspect, the present invention provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that increases the expression and / or activity of SASH1.

[0016] Suitably the agent is a small organic molecule.

[0017] Suitably, for the first, second, third and fourth aspects, the anti-cancer agent or cancer treatment is or comprises a PARP inhibitor. Preferably, the PARP inhibitor is selected from the group consisting of olaparib, veliparib, rucaparib, iniparib, talazoparib, niraparib, 3-aminobenzamide, ME0328, PJ34, AG-14361, INO-1001, UPF-1069, AZD-2461, CEP9722, A-966492, and any combination thereof.

[0018] In a fifth aspect, the present invention provides a method for identifying and / or producing an agent for use in treating cancer in a subject, comprising: (a) contacting a cell expressing a SASH1 nucleic acid or SASH1 protein with a candidate agent; (b) determining whether the candidate agent alters the expression and / or activity of SASH1.

[0019] In some embodiments, the candidate agent at least partially reduces, eliminates, suppresses, or inhibits the expression and / or activity of SASH1, hi alternative embodiments, the candidate agent at least partially increases the expression and / or activity of SASH1.

[0020] The agent is suitably an antibody or a small organic molecule.

[0021] In a sixth aspect, the invention provides an agent identified or produced by the method of the fourth aspect.

[0022] In a seventh aspect, the present invention provides a kit for predicting the responsiveness of a cancer to an anticancer drug in a subject, wherein the anticancer drug inhibits the activity of an enzyme that mediates the repair of DNA strand breaks, and the kit includes at least one reagent capable of determining the expression level of SASH1 protein or a nucleic acid encoding the SASH1 protein in one or more cancer cells, cancer tissues, or cancer organs of the subject, wherein the expression level of the SASH1 protein or the nucleic acid encoding the SASH1 protein indicates a relative increase or decrease in the responsiveness of the cancer to the anticancer drug or is correlated with a relative increase or decrease in the responsiveness of the cancer to the anticancer drug.

[0023] In particular embodiments, a relative decrease in the level of SASH1 protein or a nucleic acid encoding a SASH1 protein indicates a relative increase in the cancer's response to an anti-cancer drug or is correlated with a relative increase in the cancer's response to an anti-cancer drug, and / or a relative increase in the level of SASH1 protein or a nucleic acid encoding a SASH1 protein indicates a relative decrease in the cancer's response to an anti-cancer drug or is correlated with a relative decrease in the cancer's response to an anti-cancer drug.

[0024] In the above aspect, the enzyme is suitably poly(ADP-ribose) polymerase (PARP). Therefore, the anticancer drug is suitably a PARP inhibitor or includes a PARP inhibitor. The PARP inhibitor is preferably selected from the group consisting of olaparib, veliparib, rucaparib, iniparib, talazoparib, niraparib, 3-aminobenzamide, ME0328, PJ34, AG-14361, INO-1001, UPF-1069, AZD-2461, CEP9722, A-966492, and any combination thereof.

[0025] Suitably, the kit further comprises a collection of data comprising correlation data between the expression level of the SASH1 protein or nucleic acid encoding the SASH1 protein and the responsiveness of the cancer to an anti-cancer drug.

[0026] The collection of data suitably resides on a computer readable medium.

[0027] In particular embodiments, the kit is for use in the method of the above aspects.

[0028] Suitably, the cancer in the above aspect is a cancer of the reproductive system. Preferably, the cancer of the reproductive system includes breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, and testicular cancer. More preferably, the cancer of the reproductive system is breast cancer.

[0029] In another embodiment, the cancer of the above aspect is or comprises lung cancer, preferably including squamous cell carcinoma, adenocarcinoma, large cell carcinoma, small cell carcinoma, and mesothelioma.

[0030] In an eighth aspect, the present invention provides a method for determining the prognosis of a subject for breast cancer, comprising determining the expression level of SASH1 nucleic acid or SASH1 protein in one or more cancer cells, cancer tissues, or cancer organs of the subject, wherein the expression level of SASH1 indicates a worse or better prognosis for breast cancer, or is correlated with a worse or better prognosis for breast cancer.

[0031] In particular embodiments, the breast cancer is ER-positive (ER + ) breast cancer or ER-negative (ER - )Breast cancer.

[0032] Suitably, the subject of the above aspects is a mammal, preferably a human.

[0033] The terms "comprises," "including," and similar terms shall mean open-ended inclusion unless the context requires otherwise, and a listed list of elements or features does not include only the elements mentioned or listed, but may include other elements or features not listed or mentioned.

[0034] The indefinite article "a" or "an" is used herein to mean or include a singular or plural element or feature and should not be understood to mean or define "one" or "single" element or feature. For example, "a" cell includes one cell, one or more cells, and a plurality of cells. [Brief explanation of the drawings]

[0035] [Figure 1] Cleavage of SASH1 by caspase-3 is required for apoptosis. A) Recombinant caspase-3 cleaves SASH1 between amino acids 230 and 231. B) Overexpression of the cleaved fragment of SASH1 demonstrates that cleavage of SASH1 is required for the apoptotic function of SASH1.

[0036] [Figure 2] Overexpression of SASH1 or the C-terminal 231-1247 region of truncated SASH1 increases the levels of NF-κB nuclear protein. A) Quantification of NF-κB nuclear protein levels via immunofluorescence.

[0037] [Figure 3] SASH1 is required for homologous recombination and genome stability. A) SASH1 protein levels accumulate after a DNA-damaging stimulus. B) Reduced SASH1 function significantly reduces the efficiency of homologous recombination. C) SASH1 is required for efficient loss of the DNA damage marker yH2AX. D) Reduced SASH1 function results in genome instability as measured via the comet assay.

[0038] [Figure 4]SASH1 protein levels correlate with sensitivity to olaparib, talazoparib, and veliparib. Correlation between the IC50 of the PARP inhibitor olaparib and SASH1 protein levels in A) breast cancer cell lines, D) lung cancer cell lines, and E) ovarian cancer cell lines. B) Correlation between the IC50 of the PARP inhibitor talazoparib and SASH1 protein levels in breast cancer cell lines. C) Correlation between the IC50 of the PARP inhibitor veliparib and SASH1 protein levels in breast cancer cell lines.

[0039] [Figure 5] Loss of SASH1 function increases the sensitivity of cancer cells to olaparib. A) Cell lines with reduced SASH1 function have a reduced IC50 for olaparib. B) Combined data showing that cell lines with reduced SASH1 function have a significantly reduced IC50 for olaparib.

[0040] [Figure 6] Overexpression of SASH1 confers resistance to olaparib in U2OS cells.

[0041] [Figure 7] No correlation exists between SASH1 and the known PARP inhibitor sensitivity markers BRCA1 or BRCA2. Correlation of SASH1 mRNA expression with A) BRCA1 or B) BRCA2. Online database COXPRES.

[0042] [Figure 8]SASH1 expression is associated with recurrence and survival in breast cancer. (A) Kaplan-Meier analysis of the relationship between SASH1 mRNA (left) or SASH1 protein (right) expression and clinical outcomes in ER-positive and ER-negative breast cancers. BCSS: breast cancer-specific survival. Log-rank p-values ​​and hazard ratios (HRs; 95% confidence intervals in parentheses) are shown. (B) Representative SASH1 IHC images of breast cancer tissue microarray cores. Two grade 3 (G3) invasive ductal carcinomas (IDCs), one negative and one strongly positive for nuclear SASH1 expression, are shown at low and high magnification. (C, D) SASH1 stratification of BCSS in ER+ breast cancers revealed similar trends in both high and low proliferation index subgroups, as measured by Ki67 expression (C) or mitotic score (D). The proportion of SASH1-high and SASH1-low cases did not differ between the ER+ subgroups with large and small proliferation indices (χ2 = χ2 test).

[0043] [Figure 9] SASH1 protein expression in breast cancer cell lines. Breast cancer cell lines were analyzed for SASH1 expression by immunoblotting. A representative immunoblot is shown in (A). (B) shows quantification of SASH1 expression levels relative to β-actin. Data shown are means ± standard deviations from three independent experiments and are normalized to MCF7 in arbitrary units.

[0044] [Figure 10]Ectopic expression of SASH1 increases cell death. (A) Confirmation of SASH1 overexpression by immunoblotting. Breast cancer cell lines were transfected with an expression construct encoding a PCMV6-SASH1-GFP fusion protein or an expression construct encoding PCMV6-GFP alone, harvested 48 hours later, and lysates were prepared and subjected to SASH1 / β-actin immunoblotting. OE: overexpression. (B) SASH1 overexpression increases death in breast cancer cell lines. 48 hours after transfection, cell lines were stained with Hoechst 33342 and propidium iodide (PI) and quantified using digital fluorescence microscopy. Data shown are the mean ± standard deviation of the relative percentage of GFP-positive and PI-positive cells (dead and late apoptotic cells) from three independent experiments. Differences between SASH1-GFP and GFP control cultures were assessed using a two-tailed t-test. *p<0.05, **p<0.005.

[0045] [Figure 11] Chloropyramine increases SASH1 expression in breast cancer cell lines. (A-H) Cells were treated with 25 μM or 50 μM chloropyramine for 24 hours, after which lysates were prepared and SASH1 protein expression was analyzed using immunoblotting. Immunoblot band intensity was quantified relative to β-actin in three independent experiments. Reproducibility and significance of changes in SASH1 expression following treatment were assessed using a two-tailed t-test. * p<0.05, ** p<0.005.

[0046] [Figure 12]Chloropyramine induces a dose-dependent decrease in breast cancer cell line proliferation that is mediated by apoptosis. (A-H) Changes in the confluency of adherent breast cancer cell lines after treatment with chloropyramine. Cells were treated with chloropyramine for 96 hours, and images were acquired and digitally analyzed using light microscopy to assess confluency relative to untreated controls. (I-K) Chloropyramine induces apoptosis in breast cancer cell lines. 48 hours after treatment with chloropyramine, cells were stained with propidium iodide and Annexin V-FITC antibody conjugate and analyzed by flow cytometry. All data shown are means ± standard deviations from three independent experiments. Statistical analysis was performed using a two-tailed t-test; * p<0.05, ** p<0.005, *** p<0.0005.

[0047] [Figure 13] SASH1 loss partially rescues chloropyramine-induced apoptosis in breast cancer cell lines. (A) Cells were transduced with negative control or SASH1 esiRNA. After 72 hours, cell lysates were prepared and SASH1 expression was analyzed relative to β-actin by immunoblotting. KD: knockdown. (B-D) Cells were transduced as described above, and chloropyramine was added 24 hours posttransfection. After 96 hours of treatment, images of cultures were captured by light microscopy and digitally analyzed to assess confluency relative to untreated controls. Data shown are means ± standard deviations from three independent experiments. A t-test was used to compare the confluency of SASH1-reduced cells with that of non-reduced cells for each dose of chloropyramine; *p<0.05.

[0048] [Figure 14] (a) SASH1 protein levels in BRCA-reduced and BRCA-retaining tumors. (b) Correlation between SASH1 levels and response to treatment with rucaparib. DETAILED DESCRIPTION OF THE INVENTION

[0049] The present invention is based, at least in part, on the surprising discovery that SASH1 is a predictive biomarker for cancer treatment with PARP inhibitors. Additionally, the inventions described herein are based on the discovery that altering, and more specifically increasing, the expression of SASH1 may be an effective anti-cancer treatment. Furthermore, the present invention is based, at least in part, on the discovery that SASH1 is a prognostic marker in cancers of the reproductive system (e.g., breast cancer) as well as other solid tumors (e.g., lung cancer, gastric cancer).

[0050] In one particular aspect, the present invention relates to a method for predicting the responsiveness of a cancer to an anticancer drug in a subject, wherein the anticancer drug inhibits the activity of an enzyme that mediates the repair of DNA strand breaks, the method comprising determining the expression level of a SASH1 nucleic acid or a protein encoded by the SASH1 nucleic acid in one or more cancer cells, cancer tissues, or cancer organs of the subject, wherein the expression level of the SASH1 nucleic acid or the protein encoded by the SASH1 nucleic acid indicates or correlates with a relative increase or decrease in the responsiveness of the cancer to the anticancer drug.

[0051] Those skilled in the art will understand that the SASH1 gene comprises a nucleotide sequence encoding the protein SAM domain and SH3 domain-containing protein 1. Other names for SASH1 can include proline-glutamic acid repeat-containing protein 3, 2500002E12Rik, DJ323M4.1, KIAA0790, DJ323M4, SH3D6A, and PEPE1. Non-limiting examples of accession numbers that indicate the nucleotide sequence of the SASH1 gene or the protein encoded by the nucleotide sequence are well understood in the art and include NM_015278 and NP_056093.3 for humans. As generally used herein, "SASH1" can refer to a SASH1 nucleic acid or a protein encoded by a SASH1 nucleic acid, unless otherwise specified.

[0052] For purposes of this invention, "isolated" means material that has been removed from a natural state or that has been manipulated by man. Isolated material can be substantially or essentially free from elements that normally accompany it in its natural state, or it can be manipulated to be in an artificial state with elements that normally accompany it in its natural state. Isolated material can be in its original form, or in a chemically synthesized form, or in a recombinant form.

[0053] As used herein, a "gene" is a nucleic acid that is a structural hereditary unit of a genome, and may include one or more nucleotide sequences that encode amino acids and one or more non-coding sequences, including, but not limited to, promoters and other 5' untranslated sequences, introns, polyadenylation sequences and other 3' untranslated sequences. In most tissues, genes are nucleic acids that comprise double-stranded DNA.

[0054] The term "nucleic acid" as used herein refers to single- or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA, and autocatalytic RNA. Nucleic acids can also be DNA-RNA hybrids. Nucleic acids typically contain a nucleotide sequence containing multiple nucleotides, including A, G, C, T, and U bases. However, the nucleotide sequence can also contain other bases, such as, but not limited to, inosine, methylcytosine, methylinosine, methyladenosine, and thiouridine.

[0055] Nucleic acids also include naturally occurring (e.g., allelic) variants of SASH1, as well as "mutant" nucleic acids that include orthologous (e.g., from different species) nucleotide sequences. Preferably, nucleic acid variants share at least 70% or 75%, preferably at least 80% or 85%, and more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequences disclosed herein.

[0056] Nucleic acids also include nucleic acid fragments. A "fragment" is a section, domain, portion, or region of a nucleic acid, each of which comprises less than 100% of the nucleotide sequence. Non-limiting examples include amplification products, primers, and probes. In particular embodiments, a nucleic acid fragment is, for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980 The nucleic acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 90, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 50, 55, 60, 65, 70, 75, 86, 87, 90, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 50, 55, 60, 65, 70, 75, 87, 88, 90, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2

[0057] As used herein, a "polynucleotide" is a nucleic acid having 80 or more consecutive nucleotides, whereas an "oligonucleotide" has fewer than 80 consecutive nucleotides. A "probe" can be a single- or double-stranded oligonucleotide or polynucleotide, and is suitably labeled for the purpose of detecting complementary sequences, for example, in Northern or Southern blotting. A "primer" is typically a single-stranded oligonucleotide, preferably having 15 to 50 consecutive nucleotides. A primer anneals to a complementary nucleic acid "template" and can be extended in a template-dependent manner by the action of a DNA polymerase (e.g., Taq polymerase, RNA-dependent DNA polymerase, Sequenase™, etc.). A "template" nucleic acid is a nucleic acid used to perform nucleic acid amplification.

[0058] "Protein" refers to an amino acid polymer. The amino acids can be natural or non-natural amino acids, with D- and L-amino acids being well understood in the art. As will be appreciated by those skilled in the art, the term "protein" also encompasses phosphorylated forms of proteins (i.e., phosphoproteins) and / or glycosylated forms of proteins (i.e., glycoproteins). A "peptide" is a protein with fewer than 50 amino acids. A "polypeptide" is a protein with more than 50 amino acids.

[0059] Protein "variants" (e.g., naturally occurring (e.g., allelic) variants and orthologs (e.g., from different species) of SASH1 are also provided. Protein variants preferably share at least 70% or 75%, preferably at least 80% or 85%, and more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences of SASH1 disclosed herein or known in the art.

[0060] Fragments of proteins are also provided, including peptide fragments that contain less than 100% of the entire amino acid sequence. In particular embodiments, fragments of proteins contain, for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 950 ... It can contain 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200 consecutive amino acids.

[0061] As generally used herein, the terms "cancer," "tumor," "malignant," and "malignancy" refer to a disease or condition, or to cells or tissues associated with that disease or condition, characterized by abnormal or abnormal proliferation and / or differentiation and / or migration of cells, often accompanied by an abnormal or abnormal molecular phenotype (including one or more gene mutations or other genetic changes involved in tumorigenesis, and / or expression of tumor markers, and / or loss of tumor suppressor expression or activity, and / or abnormal expression of cell surface markers).

[0062] Cancer may include any invasive or potentially invasive cancer, tumor, or other malignancy as listed in the NCI Cancer Index at http: / / www.cancer.gov / cancertopics / alphalist, including, but not limited to, all major forms of cancer, such as sarcoma, carcinoma, lymphoma, leukemia, and blastoma. Cancer may also include, but is not limited to, breast cancer, lung cancer (including lung adenocarcinoma), cancer of the reproductive system (including ovarian, cervical, uterine, and prostate cancer), cancer of the brain and central nervous system, head and neck cancer, gastrointestinal cancer (including colon, colorectal, and stomach cancer), liver cancer, kidney cancer, skin cancer (e.g., melanoma, skin carcinoma), cancer of blood cells (including lymphatic and myelomonocytic cancers), cancer of the endocrine system (e.g., pancreatic and pituitary), and musculoskeletal cancer (including bone and soft tissue cancers).

[0063] For the foregoing and following aspects, the cancer is suitably a cancer of the reproductive system (such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, or testicular cancer). Preferably, the cancer of the reproductive system is breast cancer. To that end, those skilled in the art will recognize that breast cancer may include all invasive breast cancers and cancer subtypes known in the art (such as triple-negative breast cancer, grade 2 breast cancer, grade 3 breast cancer, lymph node positive (LN) breast cancer, and the like). + ) Breast cancer, HER2 positive (HER2 + ) Breast cancer, ER negative (ER - ) Breast cancer, ER positive (ER + ) breast cancer, etc.

[0064] In another embodiment, the cancer of the aspects disclosed herein is or includes lung cancer. For purposes herein, it will be apparent that lung cancer can include all invasive lung cancers and cancer subtypes known in the art, including non-small cell carcinoma (i.e., squamous cell carcinoma, adenocarcinoma, large cell carcinoma), small cell carcinoma, and mesothelioma.

[0065] The term "DNA strand break" includes breaks or cuts in one strand of a double-stranded DNA (single-strand breaks) and breaks or cuts in both strands of a double-stranded DNA (double-strand breaks). Such DNA strand breaks can be caused by external factors (e.g., ionizing radiation, ultraviolet light, drugs, other mutagens present in food or the environment) or internal factors (e.g., reactive oxygen species produced during metabolic processes, errors during DNA replication).

[0066] In particular embodiments, it has been demonstrated that cancer cells have a reduced or impaired ability or ability to repair DNA strand breaks, and / or have increased sensitivity or occurrence of DNA strand breaks.In this regard, it may be demonstrated that cancer cells have defects in DNA strand break repair or increased occurrence of DNA strand breaks, which at least partially contributes to the abnormal growth, differentiation, and / or migration characteristics of cancer.Therefore, such cancer cells may be particularly sensitive to the induction of DNA strand breaks within them.Some cancer cells acquire defects in one or more specific pathways or mechanisms that repair DNA strand breaks, and then become dependent on compensation mechanisms to survive.Therefore, targeting and inhibiting the compensation mechanisms in combination with inducing DNA damage can selectively kill cancer cells, but not their normal counterparts (i.e., synthetic lethality).

[0067] In this aspect, the enzyme that mediates repair of DNA strand breaks can be any known in the art, and preferably is poly(ADP-ribose) polymerase (PARP).

[0068] Poly(ADP-ribose) polymerases (PARPs) are a family of proteins generally involved in numerous cellular processes, more specifically DNA repair and programmed cell death. The PARP family includes approximately 18 proteins (e.g., PARP1, PARP2, VPARP (PARP4), tankyrase-1 and tankyrase-2 (PARP-5a or TNKS and PARP-5b or TNKS2), PARP3, PARP6, TIPARP (or "PARP7"), PARP8, PARP9, PARP10, PARP11, PARP12, PARP14, PARP15, and PARP16), which share some homology within the catalytic domain but generally have distinct cellular functions. PARP-1 and PARP-2 are considered unique members of the family in that their catalytic activity is stimulated by the generation of DNA strand breaks. In this regard, PARP is involved in DNA damage signaling through its ability to recognize and rapidly bind to single- or double-strand breaks in DNA.

[0069] In connection with the above, the anticancer drug suitably is or includes a PARP inhibitor. The term "PARP inhibitor" generally refers herein to an inhibitor or agonist of poly(ADP-ribose) polymerase activity. In particular embodiments, the PARP inhibitor specifically inhibits one specific PARP protein or multiple PARP proteins (PARP1 and / or PARP2). It will be apparent to those skilled in the art that when a PARP inhibitor is administered to a subject, the PARP activity in the subject's body is altered, more preferably reduced. Drugs that can reduce the expression level of one or more PARPs are also considered PARP inhibitors. In one embodiment, a prodrug of a PARP inhibitor is administered to a subject and converted to a compound in vivo to inhibit PARP.

[0070] The PARP inhibitor can be any type of compound. The compound can be, for example, an organic small molecule or a biological compound (such as an antibody or an enzyme). To this end, a person skilled in the art can determine whether a compound can inhibit the activity and / or expression of PARP by any means known in the art. Representative assays for evaluating the activity and / or inhibition of PARP include, for example, dot blot and BER assay. In the BER assay, the direct activity of PARP to form poly ADP-ribose chains is measured, for example, by using a radioactive assay using the tritiated substrate NAD or by using a specific antibody against the polymer chain formed by PARP activity.

[0071] It will be appreciated that the PARP inhibitor may be any known in the art, such as olaparib, veliparib, rucaparib, iniparib, talazoparib, niraparib, 3-aminobenzamide, ME0328, PJ34, AG-14361, INO-1001, UPF-1069, AZD-2461, CEP9722, A-966492, or any combination thereof.

[0072] As will be appreciated by those skilled in the art, the expression level of a SASH1 nucleic acid, or a protein encoded by a SASH1 nucleic acid, can be (i) higher, increased, or greater than, or (ii) lower, decreased, or reduced relative to the expression level in a control or reference sample, or relative to a threshold expression level. In one embodiment, an expression level can be classified as higher, increased, or greater if it exceeds the mean and / or median expression level of a reference population. In one embodiment, an expression level can be classified as lower, decreased, or reduced if it does not reach the mean and / or median expression level of the reference population. In this regard, the reference population can be a group of subjects with the same type and / or subgroup and / or stage and / or grade of cancer as the mammal for which the expression level is being determined.

[0073] As used herein, terms such as "higher," "increasing," "greater than," and the like refer to an increase in the amount or level of a SASH1 nucleic acid or SASH1 protein, for example, in a biological sample, as compared to a control or reference level or amount. The expression level of a SASH1 nucleic acid or SASH1 protein can be a relative or absolute value. In some embodiments, expression of a SASH1 nucleic acid or SASH1 protein is higher, increased, or greater if the expression level is about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400% greater, or at least about 500% greater than the expression level of a SASH1 nucleic acid or SASH1 protein in a control or reference.

[0074] As used herein, the terms "lower," "reduced," "decreased," and the like refer to a lower amount or level of a SASH1 nucleic acid or SASH1 protein, for example, in a biological sample, compared to a control or reference level or amount. The expression level of a SASH1 nucleic acid or SASH1 protein can be a relative or absolute value. In some embodiments, expression of a SASH1 nucleic acid or SASH1 protein is lower, reduced, or decreased if the expression level is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, or 0.0001% of the expression level of a SASH1 nucleic acid or SASH1 protein in a control or reference.

[0075] The term "control sample" typically refers to a biological sample from a disease-free individual who is free of cancer (healthy). In one embodiment, the control sample can be from a subject known to be cancer-free. Alternatively, the control sample can be from a subject who has recovered from cancer. The control sample can be a pooled sample, an average sample, or an individual sample. An internal control is a marker from the same biological sample being investigated.

[0076] As used herein, expression level can refer to the absolute or relative amount of the expressed nucleic acid or protein. Thus, in some embodiments, the expression level of the SASH1 gene and / or its product is compared to the expression level of a control (e.g., the expression level of one or more "housekeeping" genes and / or proteins in one or more cancer cells, tissues, or organs of a mammal).

[0077] In yet another embodiment, the expression level of the SASH1 nucleic acid, or the protein encoded by the SASH1 nucleic acid, is compared to a threshold level of expression (e.g., the level of the gene and / or protein in non-cancerous tissue). The threshold level of expression is generally a quantified expression level of SASH1. Typically, an expression level of SASH1 in a sample above or below the threshold level of expression is predictive of a particular disease state or outcome. The nature or value (if any) of the threshold level of expression will typically vary depending on the method selected for determining the expression of one or more genes or their products used to determine, for example, prognosis and / or response to anti-cancer therapy (e.g., PARP inhibitor therapy) in a mammal.

[0078] A skilled artisan can use any method known in the art for measuring gene or protein expression (such as those described herein) to determine a threshold level of SASH1 nucleic acid or protein expression in a sample that can be used to determine, for example, prognosis and / or response to anti-cancer therapy. In one embodiment, the threshold level is the mean and / or median (median or absolute value) expression level of SASH1 in a reference population, e.g., having the same cancer type, and / or subgroup, and / or stage, and / or grade as the mammal for which the expression level is being determined. In addition, the concept of a threshold level of expression should not be limited to a single value or result. In this regard, a threshold level of expression can encompass multiple threshold levels of expression that may indicate, for example, a high, medium, or low probability of response to, for example, PARP inhibitor therapy.

[0079] In one embodiment, lower levels of expression of a SASH1 nucleic acid, or a protein encoded by a SASH1 nucleic acid, indicate or correlate with a relatively increased responsiveness of the cancer to anti-cancer treatment. In an alternative embodiment, lower levels of expression of a SASH1 nucleic acid, or a protein encoded by a SASH1 nucleic acid, indicate or correlate with a relatively decreased responsiveness of the cancer to anti-cancer treatment.

[0080] In a particularly preferred embodiment, where the anti-cancer treatment is or comprises a PARP inhibitor, a relatively lower expression level of the SASH1 nucleic acid, or the protein encoded by the SASH1 nucleic acid, indicates or correlates with a relatively increased responsiveness of the cancer to the PARP inhibitor. Similarly, a higher expression level of the SASH1 nucleic acid, or the protein encoded by the SASH1 nucleic acid, indicates or correlates with a relatively decreased responsiveness of the cancer to the PARP inhibitor.

[0081] Suitably, a decreased or reduced level of SASH1 indicates or correlates with a relative increase in the responsiveness of cancer to anticancer drugs. Conversely, an increased level of SASH1 indicates or correlates with a relative decrease in the responsiveness of cancer to anticancer drugs. In a particular embodiment, a decreased level of SASH1 indicates or correlates with a relative increase in the responsiveness of cancer to PARP inhibitors, and / or an increased level of SASH1 indicates or correlates with a relative decrease in the responsiveness of cancer to PARP inhibitors. In this regard, the expression level of SASH1 is useful for predicting the sensitivity and / or resistance of a subject's cancer to PARP inhibitor therapy.

[0082] The terms "determine," "measure," "estimate," "assess," and "examine" are used interchangeably herein and can encompass any form of measurement known in the art (e.g., methods described hereinafter).

[0083] The nucleic acid (e.g., RNA, mRNA, cDNA) of SASH1 can be characterized, assessed, predicted, examined, or measured by any technique known in the art, including nucleic acid sequence amplification, nucleic acid hybridization, nucleotide sequencing, mass spectrometry, and any combination thereof.

[0084] Nucleic acid amplification techniques typically involve repeated cycles of "amplifying" a target nucleotide sequence by using a polymerase to anneal one or more primers to a "template" nucleotide sequence under appropriate conditions to synthesize a nucleotide sequence complementary to the target.Nucleic acid amplification techniques are well known to those skilled in the art, and non-limiting examples include polymerase chain reaction (PCR); strand displacement amplification (SDA); rolling circle replication (RCR); nucleic acid sequence-based amplification (NASBA), Q-beta replicase amplification; helicase-dependent amplification (HAD); loop-mediated isothermal amplification (LAMP); cleavage enzyme amplification reaction (NEAR); recombinase polymerase amplification (RPA).In general, as used herein, "amplification product" refers to a nucleic acid product produced by a nucleic acid amplification technique.

[0085] PCR includes quantitative and semi-quantitative PCR, real-time PCR, allele-specific PCR, methylation-specific PCR, asymmetric PCR, nested PCR, multiplex PCR, touchdown PCR, digital PCR, and other variations and modifications to the "basic" PCR amplification.

[0086] Nucleic acid amplification techniques can be performed using DNA or RNA extracted, isolated, or otherwise obtained from cell or tissue sources. In another embodiment, nucleic acid amplification can be performed directly on appropriately processed cell or tissue samples.

[0087] Nucleic acid hybridization typically involves hybridizing a nucleotide sequence, typically in the form of a probe, to a target nucleotide sequence under appropriate conditions, followed by detection of the hybridized probe-target nucleotide sequence. Non-limiting examples of detection methods include, but are not limited to, Northern blot, slot-blotting, in situ hybridization, and fluorescence resonance energy transfer (FRET) detection. Nucleic acid hybridization can be performed using DNA or RNA extracted, isolated, or otherwise obtained from a cell or tissue source, or can be performed directly on an appropriately processed cell or tissue sample.

[0088] It will also be appreciated that a combination of nucleic acid amplification and nucleic acid hybridization may be used.

[0089] The level of SASH1 protein can be determined, assessed, estimated, examined, or measured by any technique known in the art that can detect proteins expressed by cells or tissues, whether expressed on the cell surface or intracellularly, or extracted, isolated, or otherwise obtained from a cell or tissue source. These techniques include, but are not limited to, antibody-based detection (using one or more antibodies that bind to the protein), electrophoresis, isoelectric focusing, protein sequencing, chromatographic techniques, mass spectrometry, and combinations thereof. Antibody-based detection can include, but is not limited to, flow cytometry, ELISA, immunoblotting, immunoprecipitation, in situ hybridization, immunohistochemistry, and immunocytochemistry using fluorescently labeled antibodies that bind to SASH1. Suitable techniques can be adapted for high-throughput and / or rapid analysis, for example, using protein arrays (such as, but not limited to, TissueMicroArray™ (TMA), MSD MultiArrays™, and multi-well ELISA).

[0090] Determining the expression of SASH1 can include both determining its nucleic acid level, for example, by nucleic acid amplification and / or nucleic acid hybridization, and determining its protein level.

[0091] In some embodiments, gene expression levels of SASH1 can be indirectly assessed by measuring non-coding RNAs (e.g., miRNAs) that regulate gene expression. MicroRNAs (miRNAs or miRs) are post-transcriptional regulators that bind to complementary sequences in the 3' untranslated region (3' UTR) of target mRNA transcripts, typically resulting in gene silencing. miRNAs are short RNA molecules, averaging only 22 nucleotides in length. The human genome can encode over 1,000 miRNAs, which can target approximately 60% of mammalian genes and are abundant in many types of human cells. Each miRNA can alter the expression of hundreds of individual mRNAs. Notably, miRNAs can play multiple roles in negative regulation (e.g., transcript degradation and sequestration, translational repression) and / or positive regulation (e.g., transcriptional and translational activation). In addition, aberrant miRNA expression has been implicated in various types of cancer.

[0092] A further aspect of the invention relates to treating cancer in a subject.

[0093] In a particular aspect, cancer treatment is performed in combination with determining the expression level of SASH1 protein or a nucleic acid encoding SASH1 protein in one or more cancer cells, tissues, or organs of a subject, and cancer treatment is initiated, continued, modified, or discontinued based on the determined results. In this regard, it will be understood that the methods described herein for predicting cancer response to an anti-cancer drug (such as a PARP inhibitor) can further include administering a therapeutically effective amount of the anti-cancer drug to the mammal. In a preferred embodiment, the anti-cancer drug is administered when the expression level of SASH1 indicates or correlates with a relatively increased cancer response to the anti-cancer drug.

[0094] In some embodiments, the cancer treatment comprises administering to the subject a therapeutically effective amount of an anti-cancer agent that inhibits the activity of an enzyme that mediates the repair of DNA strand breaks.

[0095] In particular embodiments, the cancer treatment comprises administering to the subject a therapeutically effective amount of an agent that inhibits or blocks the expression and / or activity of SASH1.

[0096] In another particular aspect, the present invention provides a method for treating cancer in a subject, comprising administering a therapeutically effective amount of an agent that increases the expression and / or activity of SASH1.

[0097] Suitably, the agent is a small organic molecule. One non-limiting example of a small organic molecule is chloropyramine.

[0098] In one particular aspect, the present invention provides a method of treating cancer in a subject, comprising administering a therapeutically effective amount of an agent that inhibits or blocks SASH1 expression and / or activity in combination with an anti-cancer agent that inhibits the activity of an enzyme that mediates the repair of DNA strand breaks.

[0099] Suitably, the enzyme is poly(ADP-ribose) polymerase (PARP). Thus, the anti-cancer agent may be or include a PARP inhibitor (such as those already described herein).

[0100] As previously noted, certain cancers may exhibit a reduced or impaired ability to repair DNA strand breaks, possibly through acquired defects in one or more specific DNA strand break repair pathways, and / or may exhibit increased sensitivity to or increased incidence of DNA strand breaks. Consequently, these cancer cells may become dependent on compensatory mechanisms (e.g., SASH1) to survive ongoing DNA damage. Thus, as shown herein, targeted inhibition of SASH1, combined with the induction of DNA strand breaks in cancer cells, for example, by administration of a PARP inhibitor, can achieve selective killing of cancer cells. To this end, the genetic interaction between PARP and SASH1 can be described as synthetic lethality. Synthetic lethality between two genes generally occurs when loss of either gene alone results in survival, but simultaneous loss of both genes leads to cell death.

[0101] In particular embodiments, the agent that inhibits or prevents SASH1 expression and / or activity is administered (i) before; (ii) after; or (iii) simultaneously with the administration of an anti-cancer agent. In one embodiment, the administration of the agent that inhibits or prevents SASH1 expression and / or activity and the administration of the anti-cancer agent (either sequentially or simultaneously) results in better treatment or prevention of cancer compared to treatment or prevention in which one of the agent and the anti-cancer agent is administered without the other.

[0102] The agent is administered to the subject as a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient. In this regard, any dosage form and route of administration (e.g., those provided herein) can be used to provide the subject with the composition of the present invention.

[0103] Cancer treatments can include, but are not limited to, drug therapy, chemotherapy, antibody therapy, nucleic acid and other biomolecular therapy, radiation therapy, surgery, nutritional therapy, relaxation or meditation therapy, and other natural or holistic therapies. Generally, drugs, or biomolecules (e.g., antibodies, inhibitory nucleic acids (e.g., siRNAs)), or chemotherapeutic agents are referred to herein as "anti-cancer therapeutic agents" or "anti-cancer agents."

[0104] The method of treating cancer can be a prophylactic, preventative, or therapeutic method and is suitable for treating cancer in a mammal, particularly a human. As used herein, "treating," or "treating," or "treatment" refers to a therapeutic intervention, or course of action, or protocol that at least ameliorates the symptoms of cancer after the cancer and / or its symptoms have at least begun to develop. As used herein, "preventing," or "preventing," or "prevention" refers to a therapeutic intervention, or course of action, or protocol that is initiated before the cancer and / or cancer symptoms appear in order to arrest, inhibit, or delay the development or progression of cancer or symptoms.

[0105] The phrase "therapeutically effective amount" describes the amount of a particular agent (e.g., a PARP inhibitor) sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a composition comprising one or more agents described herein that inhibit the activity of enzymes that mediate the repair of DNA strand breaks necessary to reduce and / or alleviate and / or prevent cancer or a cancer-related disease, disorder, or condition. In some embodiments, a "therapeutically effective amount" is sufficient to reduce or eliminate symptoms of cancer. In other embodiments, a "therapeutically effective amount" is an amount sufficient to achieve a desired biological effect, e.g., an amount effective to reduce or prevent cancer growth and / or metastasis.

[0106] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce a desired result without causing substantially cytotoxic effects in the subject. The effective amount of an agent useful for reducing and / or alleviating and / or preventing cancer will depend on the subject being treated, the type and severity of any associated disease and / or disorder and / or condition (e.g., the number and location of any associated metastases), and the manner in which the therapeutic composition is administered.

[0107] The anti-cancer agent is suitably administered to a mammal as a pharmaceutical composition containing a pharmaceutically acceptable carrier, diluent, or excipient.

[0108] The term "pharmaceutically acceptable carrier, diluent, or excipient" refers to a solid or liquid filler, diluent, or encapsulating material that can be safely used in systemic administration. A wide variety of carriers known in the art can be used depending on the particular route of administration. These carriers can be selected from the group consisting of sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonic saline, salts (inorganic acid salts, such as hydrochlorides, bromides, sulfates, and the like, and organic acid salts, such as acetates, propionates, malonates, and the like), and pyrogen-free water.

[0109] One useful reference describing pharmaceutically acceptable carriers, diluents, and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co., New Jersey, USA, 1991), which is incorporated herein by reference.

[0110] Any safe route of administration can be used to provide the compositions of the present invention to a patient, including oral, rectal, parenteral, sublingual, buccal, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, inhalation, intraocular, intraperitoneal, intracerebroventricular, and transdermal routes. For example, intramuscular and subcutaneous injections are suitable for administering immunotherapeutic compositions, proteinaceous vaccines, and nucleic acid vaccines.

[0111] Dosage forms include tablets, dispersions, suspensions, injectables, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches, and the like. These dosage forms can also include injection or implantation of controlled-release devices specifically designed for this purpose, or other forms of implants modified to additionally act in this manner. Controlled release of therapeutic agents can be achieved by coating the therapeutic agent with hydrophobic polymers, including, for example, acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids, and some cellulose derivatives (e.g., hydroxypropylmethylcellulose). In addition, controlled release can be achieved using other polymer matrices, and / or liposomes, and / or microspheres.

[0112] Compositions of the present invention suitable for oral or parenteral administration can be presented as discrete units (capsules, sachets, tablets, etc.), each unit containing a predetermined amount of one or more therapeutic agents of the present invention, as a powder or granules, or as a solution or suspension in either an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Such compositions can be prepared by any method of pharmacy, but all methods include the step of combining one or more of the above-mentioned agents with a carrier that constitutes the required one or more ingredients. In general, the compositions are prepared by uniformly and intimately admixing an agent of the present invention with a liquid carrier or a finely divided solid carrier, and then, if necessary, shaping the product into the desired presentation.

[0113] The above-mentioned composition can be administered in a pharmaceutically effective amount in a manner according to the dosage form.The dosage administered to a patient in the context of the present invention should be sufficient to cause a beneficial response in the patient over a reasonable period of time.The amount of the drug administered can vary depending on the subject being treated (including the patient's age, sex, weight, and overall health) and factors that depend on the physician's judgment.

[0114] In particular embodiments, anti-cancer therapy can be aimed at inhibiting the action and / or decreasing the expression of SASH1, hi other embodiments, anti-cancer therapy can be aimed at promoting the action and / or increasing the activity and / or expression of SASH1.

[0115] In alternative embodiments, anti-cancer therapies can be directed to genes or gene products other than SASH1, for example, anti-cancer therapies can target genes or gene products known to interact directly or indirectly with SASH1 and / or genes or gene products that alter the expression of SASH1.

[0116] Thus, in some embodiments, cancer treatment involves the administration of an anti-cancer agent that inhibits the activity of an enzyme that mediates the repair of DNA strand breaks, such as poly(ADP-ribose) polymerase (PARP), as previously described herein. Preferably, the anti-cancer agent is or includes a PARP inhibitor, as previously described herein.

[0117] In a particular embodiment, the present invention provides a "companion diagnostic" for cancer treatment, which provides clinicians and others with information that SASH1 expression levels can be used to safely and / or effectively administer the cancer treatment.

[0118] Suitably, the cancer is of the type described above, but is not limited thereto. Preferably, the cancer demonstrates altered or modified expression levels of SASH1.

[0119] In yet another aspect, the invention provides a method for identifying an agent for use in treating cancer in a subject, comprising: (a) contacting a cell expressing a SASH1 nucleic acid, or a protein encoded by a SASH1 nucleic acid, with a candidate agent; (b) determining whether the candidate agent alters the expression and / or activity of SASH1.

[0120] In particular embodiments, the candidate agents at least partially reduce, eliminate, suppress, or inhibit the expression and / or activity of SASH1. Given the present disclosure, it will be apparent that such agents can be used in combination with anti-cancer agents that inhibit the activity of enzymes that mediate the repair of DNA strand breaks (such as the PARP inhibitors provided herein).

[0121] In alternative embodiments, the candidate agent at least partially increases the expression and / or activity of SASH1, and to this end, the agent may be used as a single agent, or alternatively, in combination with an additional anti-cancer agent (such as an anti-cancer agent described herein).

[0122] Suitably, the agent has or exhibits few or no significant off-target and / or non-specific effects.

[0123] Preferably, the agent is an antibody or a small organic molecule.

[0124] In embodiments involving antibody inhibitors, the antibody can be polyclonal or monoclonal, natural or recombinant. Well-known protocols applicable to the production, purification, and use of antibodies can be found, for example, in Chapter 2 of Coligan et al., "Current Protocols in Immunology" (John Wiley & Sons, New York, 1991-1994), and in Harlow, E. and Lane, D., "Antibodies: A Laboratory Manual," Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988, both of which are incorporated herein by reference.

[0125] Generally, the antibodies of the present invention bind to or form complexes with an isolated protein, fragment, variant, or derivative of the protein product of SASH1. For example, the antibody can be a polyclonal antibody. Such antibodies can be prepared, for example, by injecting an isolated protein, fragment, variant, or derivative of the protein product of SASH1 into a producing species (including mice and rabbits) to obtain polyclonal antisera. Methods for producing polyclonal antibodies are well known to those skilled in the art. Representative protocols that can be used are described, for example, in Coligan et al., "CURRENT PROTOCOLS IN IMMUNOLOGY," supra, and Harlow and Lane, 1988, supra.

[0126] Monoclonal antibodies can be produced using standard methods, for example, as described by Kohler and Milstein (1975, Nature 256:495), which are incorporated herein by reference, or according to more recent modifications thereof, for example, as described in Coligan et al., "CURRENT PROTOCOLS IN IMMUNOLOGY," supra, by immortalizing spleen or other antibody-producing cells from a production species inoculated with one or more of the isolated SASH1 protein products and / or fragments, variants and / or derivatives thereof.

[0127] Typically, the inhibitory activity of a candidate inhibitory antibody can be assessed by in vitro and / or in vivo assays that detect or measure the expression level and / or activity of SASH1 protein in the presence of the antibody.

[0128] In embodiments involving small organic molecule inhibitors, assessing inhibitory activity can involve screening large compound libraries, where hundreds of thousands to hundreds of millions of candidate inhibitors (synthetic small organic molecules or compounds including natural products, e.g., inhibitory peptides or proteins) can be screened or tested for biological activity against any one of hundreds of molecular targets to discover potential new drugs or lead compounds. Screening methods can include, but are not limited to, computer-based ("in silico") screening and high-throughput screening based on in vitro assays.

[0129] Typically, the active compounds, or "hits," from the initial screening method are then sequentially tested through a series of other in vitro and / or in vivo tests to further characterize the active compounds, and at each stage fewer and fewer "successful" compounds are selected for further testing, until one or more drug candidates are selected for testing in human clinical trials.

[0130] At the clinical level, screening of candidate drugs can include obtaining a sample from a test subject before or after exposing the test subject to a test compound. The level of SASH1 protein in the sample is then measured and analyzed to determine whether the level and / or activity of SASH1 protein has changed after exposure to the candidate drug. For example, the level of protein product in the sample can be determined by mass spectrometry, Western blot, ELISA, and / or any other suitable means known to those skilled in the art. In addition, the activity of the protein product (such as its anti-apoptotic activity) can be determined by any method known in the art, including, for example, apoptosis assays (e.g., caspase activation and / or cleavage assays, Annexin V positivity assays, chromatin morphology assays, extracellular phosphatidylserine assays, DNA fragmentation assays).

[0131] It will be understood that subjects who have been treated with a candidate agent may be routinely examined for any physiological effects that may result from the treatment. In particular, candidate agents will be evaluated for their ability to reduce the likelihood or occurrence of cancer in a subject. Alternatively, when administered to a subject previously diagnosed with cancer, candidate agents will be screened for their ability to slow or halt the progression of cancer, as well as their ability to induce recovery from the disease.

[0132] In this regard, candidate agents identified as reducing, eliminating, suppressing, or inhibiting the expression level and / or activity of SASH1 can then be administered to patients suffering from cancer or at risk of developing cancer. Where increased activity of a biomarker is at least partially responsible for the progression and / or onset of cancer, cancer can be treated and / or the risk of cancer can be reduced by administering a candidate agent that, for example, inhibits or reduces the activity and / or expression of SASH1.

[0133] In a related aspect, the invention provides an anti-cancer agent produced or identified according to the above aspect.

[0134] In yet another aspect, the present invention provides a kit for predicting cancer response to an anticancer drug in a subject, wherein the anticancer drug inhibits the activity of an enzyme that mediates the repair of DNA strand breaks, and the kit includes at least one reagent capable of determining the expression level of SASH1 protein or a nucleic acid encoding the SASH1 protein in one or more cancer cells, cancer tissues, or cancer organs of the subject, wherein the expression level of SASH1 protein or a nucleic acid encoding the SASH1 protein indicates a relative increase or decrease in the cancer's responsiveness to the anticancer drug, or is correlated with a relative increase or decrease in responsiveness.

[0135] In particular embodiments, a relative decrease in the level of SASH1 protein or a nucleic acid encoding a SASH1 protein indicates or correlates with a relative increase in the responsiveness of the cancer to an anti-cancer drug; and / or a relative decrease in the level of SASH1 protein or a nucleic acid encoding a SASH1 protein indicates or correlates with a relative decrease in the responsiveness of the cancer to an anti-cancer drug.

[0136] In this aspect, the enzyme is suitably poly(ADP-ribose) polymerase (PARP). Therefore, the anticancer drug is suitably a PARP inhibitor or comprises a PARP inhibitor. The PARP inhibitor is preferably selected from the group consisting of olaparib, veliparib, rucaparib, iniparib, talazoparib, niraparib, 3-aminobenzamide, ME0328, PJ34, AG-14361, INO-1001, UPF-1069, AZD-2461, CEP9722, A-966492, and any combination thereof.

[0137] Suitably, the kit of the present invention further comprises reference data for correlating the expression level of the SASH1 protein or the nucleic acid encoding the SASH1 protein with the response of the cancer to an anti-cancer drug.

[0138] In particular embodiments, the reference data resides on a computer-readable medium (e.g., software implementing or utilized by any one or more of the methods or functions described herein). The computer-readable medium may be included in a storage device (such as a computer's memory (e.g., a hard disk drive or solid state drive)) and preferably contains computer-readable code components that, when selectively executed by a processor, implement one or more aspects of the present invention.

[0139] In one particular embodiment, the kit can provide a "companion diagnostic" by which information regarding the expression level of SASH1 can be utilized by clinicians and others for the safe and effective administration of anti-cancer drugs.

[0140] Suitably the kit is for use in the method of the above aspects.

[0141] In a broader aspect, the present invention provides a method for determining a subject's prognosis for breast cancer, comprising determining the expression level of SASH1 nucleic acid or SASH1 protein in one or more cancer cells, cancer tissues, or cancer organs of the subject, wherein the expression level of SASH1 indicates a worse or better prognosis for the breast cancer or is correlated with a worse or better prognosis for the breast cancer.

[0142] The breast cancer in this region is ER-positive (ER + ) breast cancer, or ER-negative (ER - ) breast cancer is appropriate.

[0143] As used herein, the terms "prognosis" and "prognostic sign" include determining a prognosis, which can predict clinical outcome (with or without medical treatment), and / or select an appropriate course of treatment (or whether treatment is likely to be effective), and / or monitor current treatment and potentially change it. This can be based at least in part on determining the expression level of SASH1, which can be combined with or supplemented by determining the expression levels of additional protein biomarkers and / or other nucleic acid biomarkers. Prognosis can also include prediction, forecast, or anticipation of any lasting (i.e., permanent) physical or psychological effects of a subject's cancer after the cancer has been successfully treated or otherwise resolved. Furthermore, prognosis can include one or more of determining metastatic potential or occurrence, determining responsiveness to treatment, achieving an appropriate treatment plan, predicting the probability or likelihood of cancer recurrence after treatment, and predicting the development of resistance to established therapies (e.g., chemotherapy). It will be appreciated that a positive prognosis typically refers to a favorable clinical outcome or outlook (e.g., long-term recurrence-free survival of the subject's cancer), whereas a negative prognosis typically refers to a negative clinical outcome or outlook (e.g., recurrence or progression of the cancer).

[0144] Suitably, the method of the above aspect further comprises the step of diagnosing the subject as having a poorer or better prognosis. In one embodiment, a relative or absolute decrease in SASH1 expression is diagnostic of a poorer or worse prognosis for the subject. + In yet another embodiment, which is breast cancer, a relative or absolute increase in expression of SASH1 is diagnostic for a more favorable prognosis for the subject. - In another embodiment, which is breast cancer, a relative or absolute decrease in the expression of SASH1 is diagnostic for a poorer or worse prognosis for the subject.

[0145] In this regard, a cancer may have a relatively poor prognosis due to one or a combination of characteristics or factors, including, but not limited to, at least partial resistance to available therapies for treating the cancer; invasiveness; metastatic potential; recurrence after treatment; and low probability of patient survival. In particular embodiments, the expression level of SASH1 is indicative of a prognosis for aggressive disease, particularly a prognosis for shorter time to biochemical recurrence and / or shorter patient survival. In yet another embodiment, the expression level of SASH1 is correlated with or indicative of metastatic cancer.

[0146] It will also be appreciated that SASH1 expression levels can be used to identify patients with a worse prognosis (e.g., larger tumors and / or higher grade tumors), such that said patients may benefit from one or more anti-cancer agents added to the typical or standard anti-cancer treatment regimen for said particular patient group.

[0147] From the above, it will be appreciated that the present invention provides methods for determining a cancer prognosis in a patient and / or predicting a cancer's response to an anti-cancer drug. In particular, broad embodiments of the present invention include treating the patient after determining the cancer prognosis and / or predicting the cancer's response to an anti-cancer drug. These embodiments thus relate to utilizing information obtained regarding the cancer's prognosis and / or predicted cancer response to an anti-cancer drug to develop and implement an anti-cancer treatment plan for the patient. In a preferred embodiment, the treatment plan is personalized for a particular patient, and therefore optimized for that particular patient.

[0148] With respect to the above aspects, non-limiting examples of the term "subject" include mammals, including humans, work animals (such as horses, camels, greyhounds), farm animals (such as cattle, sheep, horses), and pet animals (such as cats and dogs). Preferably, the subject is a human.

[0149] In order that the present invention may be more fully understood and practiced, reference is made to the following non-limiting examples. [Example]

[0150] Example 1

[0151] The putative tumor suppressor SASH1 has been implicated in apoptosis and cell proliferation. Our work also demonstrated that SASH1 is required for the repair of gene breaks through the homologous recombination DNA repair pathway. PARP inhibitors are a new generation of therapeutic agents designed to kill cancer cells with defects in the homologous recombination pathway through a process known as synthetic lethality. The best drug developed to date is olaparib. Because our data implicate SASH1 in the homologous recombination pathway, we sought to determine whether cells with low SASH1 expression (thus likely having reduced homologous recombination activity) are sensitive to PARP1 inhibitors.

[0152] result

[0153] Our current work reveals a prominent role for SASH1 in apoptosis. SASH1 is cleaved by the apoptosis regulator caspase-3, and this cleavage event is required for a normal apoptotic response (Figure 1). Furthermore, chloropyramine, a drug we identified by connectivity mapping, upregulates SASH1 and enhances apoptosis in SASH1 in a UV-dependent manner (see Example 2).

[0154] NF-κB plays a central role in the induction of apoptosis after UV exposure, which involves the translocation of the NF-κB p65 subunit from the cytoplasm to the nucleus. Our data demonstrate that cleavage of SASH1 by caspase-3 is required for nuclear transport of the NF-κB p65 subunit, and ectopic expression of the cleaved form of SASH1 is sufficient to enable nuclear transport of NF-κB (Figure 2).

[0155] SASH1 was identified as a gene counter-regulated by hSSB1, which functions in double-strand DNA break repair by homologous recombination. To assess whether SASH1, like hSSB1 and BRVA1 / 2, is required for double-strand break repair, we performed a number of assays.

[0156] We assessed the responsiveness of SASH1 to DNA damage from ionizing radiation (IR) (Figure 3A). The observed response was similar to that of other DNA damage response proteins, and protein levels remained stable. We next examined the ability of cells with reduced SASH1 function to repair double-stranded DNA breaks via homologous recombination using the GFR HR reporter cell line MCF7DRGFP. This showed that loss of SASH1 reduced homologous recombination activity (Figure 3B). This impairment is similar to that observed in cells with reduced BRCA1 and BRCA2 function. Because the epigenetic marker γH2AX is an indirect indicator of double-stranded DNA breaks, DNA damage repair can be assessed through counts of the γH2AX locus. Here, we found that removal of the γH2AX locus after IR-induced DNA damage was delayed in cells with reduced SASH1 function (Figure 3C). We next used the comet assay to assess the degree of genomic instability in SASH1-deficient cells compared with mock-treated cells. SASH1 loss resulted in significantly longer comet tails (p = 0.0003), indicating that genomic instability occurred in these cells (Figure 3D). Together, these data suggest that SASH1 is crucial for DNA repair and maintaining genome integrity through the homologous recombination pathway.

[0157] PARP proteins function through the excision repair pathway in the detection and repair of single-strand DNA breaks [9, 10]. Therefore, targeting PARP1 in tumors lacking homologous recombination has been identified as a strategy to induce cancer cell death through apoptosis and necrosis. The use of PARP inhibitors as cancer treatments is being investigated in clinical trials, with at least 53 studies currently underway, typically in combination with chemotherapeutic agents (Table 1).

[0158] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0159] The success of this approach is largely based on the presence of BRCA1 and / or BRCA2 mutations or low expression of either gene in cancer cells. A "BRCA-suppressed state" has been described in solid tumors (e.g., breast, prostate, ovarian, and lung cancer). BRCA1 / 2 proteins are required for DNA double-strand break repair through homologous recombination (the same pathway in which SASH1 functions). These mutations in BRCA1 and BRCA2 often lead to a reduced ability to repair double-strand breaks

[11] . Inhibition of PARP1 prevents the resolution of single-strand damage events, ultimately leading to double-strand DNA breaks. Cells with reduced BRCA1 / 2 function are unable to repair these double-strand breaks, leading to the accumulation of DNA damage and subsequent cell death. This induction of cell death by PARP1 inhibitors in BRCA-compromised cells is often referred to as synthetic lethality.

[0160] We further demonstrated that SASH1 protein levels linearly correlated with cancer cell sensitivity to olaparib (Figure 4). Reducing SASH1 function using siRNA increased sensitivity to olaparib treatment in 9 of 12 cell lines tested, consistent with the linear correlation between SASH1 protein levels and olaparib sensitivity (Figure 5).

[0161] Additionally, we generated stable U2OS cell lines ectopically expressing recombinant SASH1 and demonstrated that overexpression of recombinant SASH1 in U2OS cell lines was sufficient to induce resistance to olaparib (Fig. 6).

[0162] Because the correlation between SASH1 and genomic stability is similar to that observed in cells with BRCA1 / 2 reduced function, our group investigated whether SASH1 mRNA levels correlated with BRCA1 / 2 transcript levels. We performed an online bioinformatic coexpression analysis of SASH1 mRNA and BRCA1 / 2 mRNA expression (Figure 7). There was no correlation between SASH1 mRNA and BRCA1 / 2 mRNA expression levels. This indicates that SASH1 is an independent marker for PARP inhibitor sensitivity. However, it should be noted that BRCA1 / 2 mutation assessment was not considered in this analysis.

[0163] Consideration

[0164] Our data clearly demonstrate that high SASH1 protein expression is associated with olaparib resistance in both breast and lung cancer cell lines. In contrast, low levels of SASH1 protein were associated with sensitivity to PARP1 inhibition. Indeed, SASH1 expression had a direct linear relationship with olaparib sensitivity, with R values ​​of approximately 0.8 in breast cancer cell lines and 0.9 in lung cancer cell lines.

[0165] To determine whether this is directly related to SASH1 protein levels, we used siRNA to reduce SASH1 function in breast cancer cell lines, and all cell lines became sensitive to olaparib, consistent with the correlation between SASH1 levels and olaparib sensitivity. Conversely, overexpression of SASH1 in U2OS cells conferred resistance to olaparib.

[0166] Olaparib was originally designed to treat tumors with reduced BRCA1 or BRCA2 function, but BRCA1 and BRCA2 have not been reliable biomarkers for sensitivity. Consistent with this, SASH1 expression directly correlated with olaparib sensitivity, but SASH1 did not correlate with BRCA1 or BRCA2 expression. This suggests that SASH1 may be a more robust marker of olaparib sensitivity.

[0167] Our data presented here are the first to identify a molecular role for SASH1 in apoptosis. They further reveal that the tumor-suppressing activity of SASH1 may occur not only through the apoptotic pathway but also through the crucial homologous recombination pathway. We demonstrate that SASH1 functions with the tumor suppressors BRCA1 and BRCA2 in the homologous recombination repair of cytotoxic double-strand DNA breaks. Because SASH1 appears to be required for homologous recombination, we investigated whether synthetic lethality could be induced in cells with low SASH1 expression using the PARP1 inhibitor olaparib. This demonstrated a highly linear relationship between SASH1 expression and olaparib sensitivity, with an R2 of approximately 0.8 in breast cancer cell lines and 0.9 in lung cancer cell lines.

[0168] conclusion

[0169] These data indicate that intratumoral SASH1 levels are a predictive biomarker for emerging PARP1 inhibitors that target DNA repair. The R2 values ​​indicate that SASH1 is a robust marker in cell line studies that may have a significant impact on patient selection for therapy with PARP1 inhibitors, such as olaparib. Furthermore, the cell line data presented herein demonstrate that simply altering the levels of this marker in cancer cells can ablate or induce olaparib sensitivity. Importantly, no other validated and reliable single gene / protein biomarkers for PARP1 inhibitor sensitivity currently exist.

[0170] References 1. Zeller C, Hinzmann B, Seitz S, Prokoph H, Burkhard-Goettges E, Fischer J, et al. SASH1: a candidate tumor suppressor gene on chromosome 6q24.3 is downregulated in breast cancer. Oncogene. 2003rd ed. 2003;22:2972-83. 2. Rimkus C, Martini M, Friederichs J, Rosenberg R, Doll D, Siewert JR, et al. Prognostic significance of downregulated expression of the candidate tumor suppressor gene SASH1 in colon cancer. Br. J. Cancer. 2006 ed. 2006;95:1419-23. 3. Koch CA, Anderson D, Moran MF, Ellis C, Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991st ed. 1991;252:668-74. 4. Chen, Chen Y, Dong LL, Zhang JS. Effects of SASH1 on lung cancer cell proliferation, apoptosis, and invasion in vitro. Tumour Biol. 2012 ed. 2012. 5. Claudio JO, Zhu YX, Benn SJ, Shukla AH, McGlade CJ, Falcioni N, et al. HACS1 encodes a novel SH3-SAM adaptor protein differentially expressed in normal and malignant hematopoietic cells. Oncogene. 2001st ed. 2001;20:5373-7. 6. Meng Q, Zheng M, Liu H, Song C, Zhang W, Yan J, et al. SASH1 regulates proliferation, apoptosis, and invasion of osteosarcoma cell. Mol. Cell. Biochem. 2013;373:201-10. 7. Lin S, Zhang J, Xu J, Wang H, Sang Q, Xing Q, et al. Effects of SASH1 on melanoma cell proliferation and apoptosis in vitro. Mol Med Rep. 2012;6:1243-8. 8. Yang, Liu M, Gu Z, Chen J, Yan Y, Li J. Overexpression of SASH1 related to the decreased invasion ability of human glioma U251 cells. Tumour Biol. 2012 ed. 2012. 9. Marchetti C, Imperiale L, Gasparri ML, Palaia I, Pignata S, Boni T, et al. Olaparib, PARP1 inhibitor in ovarian cancer. Expert Opin Investig Drugs. 2012;21:1575-84. 10. Wang Z, Wang F, Tang T, Guo C. The role of PARP1 in the DNA damage response and its application in tumor therapy. Front. Med. SP Higher Education Press; 2012;6:156-64. 11. Savage KI, Harkin DP. BRCA1, a “complex” protein involved in the maintenance of genomic stability. FEBS J. 2015;282:630-46.

[0171] Example 2

[0172] In this study, we analyzed the relationship between SASH1 expression (both mRNA and protein) and breast cancer clinicopathological parameters. Using in silico connectivity mapping and in vitro modeling, we identified the antihistamine chloropyramine as a SASH1-dependent cell death inducer in a panel of breast cancer cell lines, suggesting that further research into its potential clinical application is warranted.

[0173] Materials and Methods

[0174] In silico analysis of the significance of SASH1 mRNA as a prognostic predictor: In multivariate models, the relationship between SASH1 mRNA expression and recurrence-free and overall survival, as well as the significance of SASH1 mRNA expression as a prognostic predictor, were analyzed using the KM Plotter breast cancer database

[13] . Three different SASH1 array probes were analyzed. Representative data from the "JetSet" optimal probes are presented

[29] (Figure 8).

[0175] Immunohistochemistry (IHC) and tissue microarray (TMA) analysis: SASH1 protein expression in breast cancer was examined by IHC analysis in the Queensland Breast Cancer Follow-Up (QFU) resource, which contains 449 invasive breast cancer cases (sampled in duplicate) and associated clinical data, including survival outcomes over 20 years

[30] . The use of these patient data and clinical samples in this study was approved by the Human Research Ethics Committees of the University of Queensland and the Royal Brisbane and Women's Hospital (RBWH).

[0176] Four-micrometer TMA sections were treated with EDTA buffer (pH 8.8) in a Decloaca apparatus for 15 minutes for antigen retrieval, followed by IHC using an anti-SASH1 antibody (Sigma Prestige, HPA029947; 1:850) and the Mach1 Universal HRP-Polymer Detection Kit (Biocare Medical). Sections were then counterstained with hematoxylin and mounted and scanned at 40x magnification on an Aperio AT Turbo slide scanner (Leica Biosystems). An experienced pathologist (AMM) scored digital images of individual tissue cores according to the intensity of tumor cell nuclei and cytoplasm and the percentage of stained tumor cells. For each case, the maximum score of duplicate tissue cores was used to determine the relationship between SASH1 expression and clinicopathological variables using χ2. 2The results were examined using the log-rank test and log-rank test (GraphPad Prism v6).

[0177] Cell culture and transfection: Breast cancer cell lines were cultured in RPMI containing 10% FCS (MDA-MB-231, MDA-MB-361, T47-D, BT-549), DMEM containing 10% FCS (MCF7, MDA-MB-468, Hs578T), or Ham's F12 containing 5% FBS and 10 μg / ml recombinant human epidermal growth factor (SUM1315) at 37°C in 5% CO2. MCF7, Hs578T, BT-549, T47-D, and SUM1315 cell lines were supplemented with 0.01 mg / ml insulin. Cells were routinely passaged using trypsin and maintained at low passage. Chloropyramine (Sigma-Aldrich) was added to adherent cultures 24 hours after plating at the indicated concentrations (0–100 μM). Cell characterization was performed by Kerry Richard at QIMR Berghofer. SPR profiles were matched against Children's Socology group cell culture and Xenograft repository for cell lines (http: / / www.cogcell.org) STR genotyping data (February 2016).

[0178] For siRNA experiments, esiRNA (Sigma) targeting SASH1 or a nonspecific control oligo was transfected using RNAiMax (Invitrogen) according to the manufacturer's instructions. Dual transfections were performed 24 hours apart, and samples were analyzed 72 hours after the first transfection to observe optimal SASH1 function reduction. For overexpression studies, full-length SASH1 cDNA was cloned into the mammalian expression vector PCMV6 (Origene). Cells were transfected in T25 flasks with 3 μg of DNA (SASH1-GFP or GFP) and 6 μl of Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were harvested 24–48 hours after transfection to assess optimal overexpression and death, as indicated in the figure legends.

[0179] Immunoblotting: Immunoblotting was performed as previously described

[31] . Briefly, cells were lysed (20 mM Hepes pH 8.0, 150 mM KCl, 5% glycerol, 10 mM MgCl2, 0.5 mM EDTA, 0.02% NP-40, freshly supplemented NaF, NaVO4, PMSF, and protease inhibitors) and then sonicated. The lysate was cleared by centrifugation, and protein concentration was estimated using a Bradford assay (Bo-Rad). Typically, 50 μg of protein lysate was resolved on a Bolt 4-12% gradient gel (Invitrogen), and the proteins were then transferred to a nitrocellulose membrane (Bo-Rad). The membrane was blocked for 1 hour in PBS containing 2% fish skin gelatin and 1% Tween-20 (Sigma) and then incubated overnight at 4°C with primary antibodies in the same buffer. After incubation with secondary antibodies, membranes were visualized using a Li-COR Odyssey infrared scanner, and fluorescence intensity was quantified relative to loading controls (β-actin or histone H3) using Image J software.

[0180] Cell death assay: After incubating cells with the indicated treatments, propidium iodide (10 μg / ml) and Hoechst (1 μg / ml) were added 30 minutes before imaging. Cell images were acquired on an IN Cell Analyzer 2200 (GE Healthcare; 10x objective). Live / dead cell analysis was performed using InCell Analysis software.

[0181] Cell confluence assay: Cells were seeded at 2,500 cells per well in a 96-well plate (Nunc). After allowing cells to adhere for 24 hours, chloropyramine was added and images were acquired every 2 hours for 96 hours using an IncuCyte ZOOM® Viable Cell Imager to calculate confluence.

[0182] Annexin V / propidium iodide (PI) analysis: Annexin V / PI staining was performed as described

[32] . Briefly, treated or untreated cells (adherent and floating cells) were harvested using trypsin and centrifugation, washed in PBS, and stained according to the protocol of the Promega Annexin V-FITC Apoptosis Detection Kit. Annexin V-positive (apoptotic) cells were detected using a Gallios flow cytometry system and quantified using Flow Jo software.

[0183] Statistical analysis: Most statistical tests were performed using Graph Pad Prism V6. 2The association between SASH1 expression in breast cancer and clinicopathological variables was examined using a Kaplan-Meier test. The relationship between SASH1 expression in breast cancer and recurrence-free or overall survival was depicted using Kaplan-Meier curves and analyzed using the log-rank test. Analysis of SASH1-mediated changes in apoptosis and proliferation, and SASH1 expression after chloropyramine treatment was examined using a two-tailed Student's t-test. Multivariate analysis of the significance of SASH1 mRNA as a prognostic predictor was performed using available parameters (MKI67 and ERBB2 expression) and built-in functions in the KM plotter database

[13] .

[0184] ER + For multivariate analysis of the significance of SASH1 protein as a prognostic predictor in breast cancer, a stepwise Cox regression analysis was performed using MedCalc® software (v13.2) including HER2 status (determined by CISH according to diagnostic criteria), Ki67 status (nuclear staining in at least 20% of tumor cells), histological grade (assessed by experienced pathologists (SRL)), and tumor size (derived from clinicopathological reports). These data were compiled from 223 ER cases. + In all cases, the data were complete. A p value of less than 0.05 was considered significant.

[0185] Drug screening using connectivity mapping: We employed a gene expression connectivity mapping approach to identify candidate compounds that could induce SASH1 expression. SASH1 was mapped to the Affimetrix HG-U133A probe set to form a query gene signature, which was compared to the expression profiles of reference drugs in the CMap02 database using the sscMap algorithm [33, 34]. Compounds with statistically significant positive connectivity to the query gene signature were selected as candidate SASH1 inducers for further laboratory validation as described.

[0186] result

[0187] We assessed the prognostic significance of SASH1 expression at the mRNA level by meta-analyzing publicly available breast cancer gene expression data combined with relevant clinical follow-up information from the KM Plotter database

[13] . Kaplan-Meier analysis revealed that loss of SASH1 mRNA expression was associated with poor prognosis in the entire patient cohort. However, dividing the large KM Plotter cohort into subgroups according to ER status revealed that the significance of SASH1 mRNA as a prognostic predictor was context-dependent. At 15 years after diagnosis, higher levels of SASH1 expression were associated with better outcome in patients with ER-positive breast cancer (p = 0.001; HR 1.4 (1.15-1.73)), but significantly worse outcome in ER-negative disease (p = 0.001; HR 0.5 (0.37-0.78)) (Figure 8A and Table 2).

[0188] The relationship between SASH1 protein expression and clinicopathological parameters was evaluated by immunohistochemistry (IHC) analysis of tissue microarrays (TMAs) in an independent cohort of 379 clinically annotated invasive breast cancers [14, 15]. Using a well-characterized anti-SASH1 antibody, widespread nuclear staining of varying intensity was observed in breast cancer cells, which was scored as negative, weakly / moderately positive, or strongly positive (Figure 8B). The proportion of stained nuclei in tumor cells was reasonably uniform within individual tissue cores. χ 2 Analysis showed that nuclear SASH1 expression was associated with ER expression but not with any other available clinicopathological parameters (Table 3; p=0.0035).

[0189] Consistent with the KM plotter data, there was strong positive SASH1 expression in the nucleus and ER. + A significant association was found between ER and favorable breast cancer-specific survival (BCSS) in breast cancer (p=0.0012; HR 1.4 [1.16-2.69]). +SASH1 stratification of outcome in breast cancer revealed similar trends in subgroups with high and low proliferation indices, based on Ki67 expression or mitotic score. The proportion of SASH1-high and SASH1-low cases was significantly higher in patients with proliferatively active ER. + Subgroups or ER with low proliferative activity + The results were similar in the ER subgroups (Fig. 8C / D). + A multivariate Cox regression model including HER2 status, Ki67 status, tumor size, and histologic grade in ER-negative cases revealed that SASH1 expression was independently associated with BCSS (HR = 0.45; 95% CI 0.27-0.77; p = 0.0037; Table 4). The trend in the ER-negative cohort was similar to the KM analysis of SASH1 mRNA data, although this did not reach statistical significance (p = 0.16). Cytoplasmic staining of tumor cells was present in approximately 5% of cases (n = 19 / 379), but there was no association with any of the available clinicopathologic parameters examined, including survival.

[0190] To explore the function of SASH1 in breast cancer, we quantified its protein expression in three ER-positive and five ER-negative breast cancer cell lines by immunoblot analysis. The results revealed variable expression among the cell lines, with high expression in three cell lines (T47-D, BT-549, and MDA-MB-231), intermediate expression in two cell lines (Hs578T and SUM-315), and low expression in three cell lines (MCF7, MDA-MB-361, and MDA-MB-468) (Fig. 9A and 9B). There was no clear correlation between SASH1 expression and ER status in this panel of cell lines.

[0191] SASH1 has previously been described as a tumor suppressor, and its overexpression increased cell death in lung cancer, melanoma, osteosarcoma, and glioma cell lines [5, 10-12]. To investigate this, we transiently overexpressed a SASH1-GFP fusion protein in breast cancer cell lines. Overexpression resulted in cell death in seven of eight cell lines tested, with significant cell death in five, and only MCF7 cells, which have reduced caspase-3 function, showed no response (Figure 10).

[0192] We hypothesized that increasing SASH1 levels may represent a novel approach for cancer therapy, and used connectivity screening with the cmap database (Broad Institute

[16] ) to identify drugs that lead to SASH1 induction. In doing so, we identified a direct correlation between chlorpyramine treatment and SASH1 mRNA expression (p = 0.000005, z score 2.431). Chlorpyramine is a first-generation reversible H1 receptor antagonist approved in several European countries for the management of allergic conditions (e.g., conjunctivitis and bronchial asthma).

[0193] After confirming chloropyramine-mediated SASH1 induction at the protein level in breast cancer cell lines (Figure 11), we investigated whether this treatment could mimic the effects of SASH1 overexpression on cell proliferation and survival. Treatment with chloropyramine inhibited cell proliferation in seven of the eight treated lines (Figures 12A-H). To determine whether this was due to the induction of apoptosis, we analyzed the levels of annexin V after treatment in the three most sensitive cell lines, T47-D, MDA-MB-231, and BT-549. All three lines showed an increase in annexin V (Figures 12I-K), indicating the induction of apoptosis.

[0194] To determine whether chloropyramine-induced cell death is dependent on SASH1, we transfected and then treated T47-D, MDA-MB-231, and BT-549 cells with siRNA targeting SASH1. These experiments demonstrated that loss of SASH1 function partially rescued the cell death response in all three systems (Figures 13A-D). This suggests that chloropyramine-induced cell death is at least partially dependent on SASH1 function.

[0195] [Table 2]

[0196] [Table 3-1] [Table 3-2]

[0197] [Table 4]

[0198] Consideration

[0199] Based on its association with favorable prognosis in several human malignancies [4-6, 10-12, 19, 20] and evidence of deleterious effects on cancer cell line viability in vitro [5, 10-12], SASH1 has been proposed to be a tumor suppressor [4-7, 10-12, 21, 22]. While mechanistic studies are generally lacking to date, some evidence exists suggesting that SASH1 can inhibit PI3K and Akt signaling

[23] . Because its significance as a prognostic predictor in breast cancer has been less well characterized than in other malignancies, we investigated this using two well-annotated clinical sample cohorts. Overall, we found that SASH1 was associated with favorable prognosis, but stratifying these cases based on ER status revealed that this was dominated by a higher prevalence of ER-positive cases (75-80% of the analyzed cohort). Indeed, SASH1 was associated with poor outcome in ER-negative breast cancer. Although SASH1 has been called a tumor suppressor, contradictory data from ER-positive and ER-negative breast cancers suggest that this may be an oversimplification of the role of SASH1 and that context is crucial. Indeed, we examined other KM Plotter cancer datasets and found a strong association between SASH1 mRNA expression and better overall survival in lung cancer but poor outcome in gastric cancer (data not shown).

[0200] In this study, we found no clear correlation between SASH1 expression and ER status in a panel of eight breast cancer cell lines. Ectopic expression of SASH1 reduced cell viability independently of ER status. These observations, taken together, suggest that in some contexts, SASH1 suppression may reflect a process necessary for cancer cell survival and invasive clinical behavior. Therefore, we hypothesized that increasing SASH1 expression could represent a novel therapeutic strategy. To identify drug candidates with this capability, we performed in silico connectivity mapping using the cmap database (Broad Institute

[16] ) and identified the antihistamine chloropyramine as a candidate SASH1 inducer. Other researchers have shown that chloropyramine reduces the viability of cell lines from melanoma, neuroblastoma, breast cancer, and pancreatic cancer, possibly through the suppression of FAK signaling and VEGFR3 signaling [17, 18, 24-26]. Consistent with the connectivity screen, chloropyramine induced SASH1 expression in seven of eight breast cancer cell lines examined and reduced viability in six of the eight. Transduction of the three most sensitive cell lines with SASH1 siRNA prior to treatment partially rescued the cytotoxic response, suggesting that chloropyramine-induced cancer cell line death is mediated, at least in part, by SASH1. At least one report suggests that chloropyramine can reduce breast cancer xenograft growth in vivo

[18] , but additional preclinical studies are needed to more comprehensively characterize the agent's antitumor activity.

[0201] Although breast cancer management has improved over the past few decades, 15–20% of patients in Australia, the United States, and the UK still do not survive 10 years after diagnosis [27, 28]. This accounts for a significant proportion of cancer-related morbidity and mortality, as well as a significant cost to public health. Tumors that do not respond to current first-line treatments are likely to be more complex and multimodal. Future disease control will depend on an increased understanding of the disease's molecular biology, leading to the identification of novel personalized medicine therapeutic approaches coupled with companion diagnostics. In silico connectivity screening provides a rapid means for gene-drug relationship identification and repositioning. In silico mapping of the relationship between SASH1 induction and "off-the-shelf" drugs identified chloropyramine as a novel candidate with in vitro antitumor activity. Chlorpyramine and other first-generation H1 antagonists are sedatives due to their ability to cross the blood-brain barrier and have therefore been replaced by peripherally acting agents in the treatment of allergies. Because brain uptake may be desirable in molecular oncology, and chloropyramine is otherwise well tolerated, the potential use of chloropyramine or structurally related agents for the low-toxicity treatment of breast and other cancers merits further investigation through mechanistic and preclinical studies. Furthermore, biomarker-based phase 0 and dose-finding phase 1 neoadjuvant clinical trials in breast cancer patients may confirm the pharmacodynamic induction of SASH1 by chloropyramine, which could provide a rationale for further future repositioning studies of this agent. This is particularly important in triple-negative breast cancer, where treatment options are currently limited, as well as in ER- and HER2-positive breast cancers, which have become resistant to current therapeutic approaches.

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[0203] Example 3

[0204] The study investigated the relationship between SASH1 protein levels and the sensitivity of ovarian cancer tumor samples to PARP inhibitors using PDX tumor samples from ovarian cancer patients.

[0205] Materials and Methods

[0206] PDX tissue from 12 patients with high-grade ovarian cancer and known response to rucaparib was received from the Walter and Eliza Hall Institute. These samples are described in more detail in Kondrashova et al. [1]. 15 mg of tissue from each sample was lysed in 400 μl of RIPA buffer containing protease and phosphatase inhibitors (Invitrogen). Samples were homogenized, sonicated, and centrifugation removed cellular debris. 40 μg was run on a gel, a BCA assay was performed to calculate protein concentration, and Western blots were performed as previously described. SASH1 protein levels were quantified using ImageJ and normalized to γ-tubulin.

[0207] result

[0208] Although SASH1 protein levels are generally lower in BRCA-reduced tumors and higher in BRCA-retaining tumors, there remains considerable overlap in SASH1 levels between these two groups (Figure 14A). In contrast, SASH1 levels provided a much stronger and more significant predictor of response to treatment with rucaparib, with lower SASH1 levels found in sensitive or mixed-response patients and higher SASH1 levels found in refractory patients (Figure 14B).

[0209] Consideration

[0210] These results demonstrate that SASH1 levels have the ability to predict patient and tumor sensitivity to PARP inhibitors, and such information may be useful for stratifying patients into treatment groups to improve clinical outcomes. These results also suggest that measuring SASH1 levels in patients could significantly improve the efficiency and reduce the costs of conducting clinical trials to develop new PARP inhibitors by enabling clinicians to identify patients who are more likely to respond to PARP inhibitors.

[0211] References 1. Olga Kondrashova, Monique Topp, et al,. Methylation of all BRCA1 copies predicts response to the PARP inhibitor rucaparib in ovarian carcinoma. Nature Communications volume 9, Article number: 3970 (2018).

[0212] The objective throughout this specification has been to describe preferred embodiments of the invention without limiting the invention to any one particular embodiment or particular collection of features. Accordingly, those skilled in the art will appreciate, in light of this disclosure, that various changes and modifications can be made to the particular embodiments exemplified without departing from the scope of the invention.

[0213] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

Claims

1. A method for predicting the responsiveness of a cancer to an anticancer drug in a subject, wherein the anticancer drug inhibits the activity of an enzyme that mediates the repair of DNA strand breaks, the method comprising a step of determining the expression level of SASH1 nucleic acid or SASH1 protein in one or more cancer cells, cancer tissues, or cancer organs of the subject, wherein the expression level of the SASH1 nucleic acid or SASH1 protein indicates a relative increase or decrease in the responsiveness of the cancer to the anticancer drug or is correlated with a relative increase or decrease in the responsiveness of the cancer to the anticancer drug.

2. 2. The method of claim 1, wherein the enzyme is poly(ADP-ribose) polymerase (PARP).

3. 3. The method of claim 1 or 2, wherein the anticancer agent is or comprises a PARP inhibitor.

4. 4. The method of claim 3, wherein the PARP inhibitor is selected from the group consisting of olaparib, veliparib, rucaparib, iniparib, talazoparib, niraparib, 3-aminobenzamide, ME0328, PJ34, AG-14361, INO-1001, UPF-1069, AZD-2461, CEP9722, A-966492, and any combination thereof.

5. The method of claim 3 or 4, wherein a decrease in the level of SASH1 nucleic acid or SASH1 protein indicates a relative increase in the response of the cancer to the PARP inhibitor or is correlated with a relative increase in the response of the cancer to the PARP inhibitor, and / or an increase in the level of SASH1 nucleic acid or SASH1 protein indicates a relative decrease in the response of the cancer to the PARP inhibitor or is correlated with a relative decrease in the response of the cancer to the PARP inhibitor.

6. 6. The method of any one of claims 1 to 5, further comprising treating said cancer in said subject.

7. A method for treating cancer in a subject, comprising determining the expression level of SASH1 nucleic acid or SASH1 protein in one or more cancer cells, cancer tissues, or cancer organs of the subject, and initiating, continuing, modifying, or discontinuing cancer treatment based on the results.

8. 8. The method of claim 7, wherein the cancer treatment comprises administering a therapeutically effective amount of an anti-cancer agent that inhibits the activity of an enzyme that mediates repair of DNA strand breaks.

9. The method of claim 7 or 8, wherein the cancer treatment comprises administering to the subject a therapeutically effective amount of a drug that inhibits or blocks the expression and / or activity of SASH1.

10. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an agent that inhibits or blocks the expression and / or activity of SASH1 in combination with an anticancer agent that inhibits the activity of an enzyme that mediates the repair of DNA strand breaks.

11. The method of any one of claims 8 to 10, wherein the enzyme is poly(ADP-ribose) polymerase (PARP).

12. The method of any one of claims 8 to 11, wherein the anticancer agent is or comprises a PARP inhibitor.

13. 13. The method of claim 12, wherein the PARP inhibitor is selected from the group consisting of olaparib, veliparib, rucaparib, iniparib, talazoparib, niraparib, 3-aminobenzamide, ME0328, PJ34, AG-14361, INO-1001, UPF-1069, AZD-2461, CEP9722, A-966492, and any combination thereof.

14. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an agent that increases the expression and / or activity of SASH1.

15. 15. The method of claim 14, wherein the agent is a small organic molecule.

16. 1. A method of identifying and / or producing an agent for use in treating cancer in a subject, comprising: (a) contacting a cell expressing a SASH1 protein or a SASH1 nucleic acid with a candidate agent; (b) determining whether the candidate agent alters the expression and / or activity of SASH1.

17. The method of claim 16, wherein the candidate agent at least partially reduces, eliminates, suppresses or inhibits the expression and / or activity of SASH1.

18. The method of claim 16, wherein the candidate agent at least partially increases the expression and / or activity of SASH1.

19. The method of any one of claims 16 to 18, wherein the candidate agent is an antibody or a small organic molecule.

20. 20. The method of any one of claims 16 to 19, wherein the candidate agent is suitable for use in the treatment of cancer.

21. 21. The method of any one of claims 1 to 20, wherein the cancer is a cancer of the reproductive system.

22. 22. The method of claim 21, wherein said cancer of the reproductive system comprises breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, or testicular cancer.

23. 23. The method of claim 22, wherein said cancer of the reproductive system is breast cancer.

24. 21. The method of any one of claims 1 to 20, wherein the cancer is or comprises lung cancer.

25. A drug for treating cancer, produced or identified by the method of any one of claims 15 to 24.

26. 26. The agent of claim 25 for use in accordance with the method of any one of claims 6 to 15 or 21 to 24.

27. A kit for predicting the responsiveness of a cancer to an anticancer drug in a subject, wherein the anticancer drug inhibits the activity of an enzyme that mediates the repair of DNA strand breaks, and the kit includes at least one reagent capable of determining the expression level of SASH1 protein or a nucleic acid encoding the SASH1 protein in one or more cancer cells, cancer tissues, or cancer organs of the subject, wherein the expression level of the SASH1 protein or the nucleic acid encoding the SASH1 protein indicates a relative increase or decrease in the responsiveness of the cancer to the anticancer drug or is correlated with a relative increase or decrease in the responsiveness of the cancer to the anticancer drug.

28. A kit according to claim 27 for use in a method according to any one of claims 1 to 15.

29. A method for determining the prognosis of a subject for breast cancer, comprising a step of determining the expression level of SASH1 nucleic acid or SASH1 protein in one or more cancer cells, cancer tissues, or cancer organs of the subject, wherein an altered expression level of SASH1 indicates a worse or better prognosis for the breast cancer, or is correlated with a worse or better prognosis for the breast cancer.

30. The breast cancer is ER-positive (ER + ) breast cancer or ER-negative (ER - 30. The method of claim 29, wherein the cancer is breast cancer.

31. The method of any one of claims 1 to 24, 29, and 30, wherein the subject is a human.

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