WO2021030783A1 - Compositions and methods for the treatment cancer and cns disorders - Google Patents

Abstract

The present disclosure provides compositions and methods for the treatment of cancer and central nervous system disorders, downregulating glutamate export in a cell or preventing and/or reducing neuron cell death in a subject. In one aspect, the present disclosure provides a method of downregulating glutamate export in a cell, the method comprising administering to the subject a therapeutically effective amount of an ABL inhibitor and/or a SLC7A 11 inhibitor such that the glutamate export is downregulated in the cell.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF CANCER AND

CNS DISORDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/887,045, filed August 15, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

FEDERAL FUNDING LEGEND

[0002] This invention was made with Government support under Federal Grant Nos. R01 CA 195549 awarded by the National Institutes of Health. The Federal Government has certain rights to this invention.

BACKGROUND

Field of the Invention

[0003] The present disclosure provides compositions and methods for the treatment of cancer and central nervous system (“CNS”) disorders.

Description of Related Art

[0004] The ABL family of non-receptor tyrosine kinases are essential for proper embiyonic development. Kinase activity is dampened following birth; however, in the presence of injury or during cancer progression the activity levels of the kinases increase. Emerging studies have shown a role for the ABL family kinases, ABL1 and ABL2, in the progression of solid tumors and metastasis. Recent work has also shown that activation of ABL kinases in pathological conditions promotes neurodegeneration, and that treatment with ABL kinase inhibitors improves astrocytic and synaptic function and reverses cognitive and motor decline in preclinical mouse models.

[0005] It was previously reported that ABL1 and ABL2 are paralogues which have both overlapping and divergent functions. Wang J., et al. (2015). For example, ABL2 is highly expressed in the brain, while ABL1 is ubiquitously expressed. Id. However, ABL kinases are downregulated in postnatal mice, and ABL1 and ABL2 are both hyperactive in metastatic tumors and in response to injury in the adult. Id.

[0006] Activation of ABL kinases in solid tumors is driven by enhanced ABL1 or ABL2 expression and/or activation due to amplification, increased gene expression, enhanced protein expression, and/or increased enzymatic activity in response to stimulation by oncogenic tyrosine kinases, chemokine receptors, oxidative stress, metabolic stress, and/or inactivation of negative regulatory proteins. Sequencing projects report ABL amplification, somatic mutations and/or increased mRNA expression have been shown in multiple types of solid tumors, including liver, uterine, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma and kidney renal clear cell carcinoma (www.cbioportal.org). These findings are consistent with reports of elevated ABL2 expression in advanced high-grade breast, colorectal, pancreatic, renal and gastric tumors.

[0007] With an estimated 234,000 new cases and 154,000 deaths in 2018, lung cancer is the leading cause of cancer deaths in the United States, accounting for one-quarter of all cancer deaths. Approximately, 80% of deaths are associated with smoking, which confers a 25-fold increase in relative risk. Smoking-associated lung cancer has one of the highest mutational burdens of all cancers. Despite advancements in molecularly targeted therapies for patients harboring actionable genetic abnormalities such as mutations in EGFR, ALK, RET, or BRAF, the majority of lung cancers lack identifiable driver oncogenes or harbor mutations in KRAS, TP53, or other clinically inactionable genetic abnormalities.

[0008] Furthermore, lung cancer patients exhibit the highest prevalence (~50%) of brain metastasis across all cancer types. Bamholtz-Sloan et al. (2004); Schouten et al. (2002). In contrast to other primary tumors, 10%-20% of lung cancer patients present with brain metastases at the time of diagnosis. Nayak et al. (2012); Schuette (2004); Shin et al. (2014). Patients with lung adenocarcinoma, a subtype of non-small cell lung cancer (NSCLC), represent the largest group of patients with brain-metastatic disease. Lung adenocarcinomas driven by mutations in the epidermal growth factor receptor (EGFR) are at particularly high risk for developing brain metastases. Shin et al. (2014).

[0009] Preclinical studies in mouse models have identified genes that mediate metastasis to the brain. Chen et al. (2016); Er et al. (2018); Sevenich et al. (2014); Valiente et al. (2014).

Unfortunately, therapies targeting these and other metastasis regulators have not translated into effective therapies against brain metastasis. The ABL family of tyrosine kinases, ABLl and ABL2, promotes lung cancer metastasis to multiple organ sites in part through stabilization of the transcriptional co-activator TAZ (encoded by WWTR1 ). Hoi et al. (2019); Gu et al. (2016). Upon nuclear translocation, TAZ binds to the TEAD family of transcription factors to coordinate expression of target genes implicated in organ size (Yu et al., (2015)), sternness (Kim et al., (2015)), cell migration (Feng et al., (2016)), and epithelial-to- mesenchymal transition (“EMT”) (Moroishi et al., (2015)). [0010] Despite early clinical successes with next-generation blood-brain barrier (BBB)- penetrant EGFR tyrosine kinase inhibitors (TKIs) such as osimertinib, relapses for lung adenocarcinoma patients with intracranial disease remain the rule rather than the exception. Kelly et al. (2018); Oxnard et al. (2018). Thus, the lack of durable treatment options for lung cancer patients suffering from brain metastases necessitates the study and discovery of novel therapeutic strategies.

[0011] Thus, there is a need for improved treatments for cancer and CNS disorders. The work described herein identifies a new role for ABL kinases in regulating SLC7A11 expression, and a novel discovery that inhibition of ABL kinases and/or SLC7A11 (1) downregulate glutamate export in a cell, (2) treats CNS disorders in a subject, (3) prevents or reduces neuronal cell death in a subject through decreasing system Xc- activity, and (4) treats cancer in a subject.

BRIEF SUMMARY OF THE DISCLOSURE

[0012] This summary is provided to introduce a selection of concepts that are further described below in the detailed description. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0013] In one aspect, the present disclosure provides a method of downregulating glutamate export in a cell, the method comprising administering to the subject a therapeutically effective amount of an ABL inhibitor and/or a SLC7A11 inhibitor such that the glutamate export is downregulated in the cell.

[0014] In a second aspect, the present disclosure provides a method of treating a central nervous system (CNS) disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of an ABL inhibitor and/or a SLC7A11 inhibitor such that the central nervous system disorder is treated in the subject.

[0015] In a third aspect, the present disclosure provides a method of preventing and/or reducing neuron cell death in a subject, the method comprising administering to the subject a therapeutically effective amount of an ABL inhibitor and/or a SLC7A11 inhibitor such that the neuron cell death in the subject is prevented and/or reduced.

[0016] In a fourth aspect, the present disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an ABL inhibitor and/or a SLC7A11 inhibitor such that the cancer is treated in the cell. [0017] In a fifth aspect, the present disclosure provides a method of treating a metastatic cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an ABL kinase inhibitor and/or a SLC7A11 inhibitor such that the metastatic cancer is treated in the cell.

[0018] In a sixth aspect, the present disclosure provides a method of treating a brain cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an ABL kinase inhibitor and/or a SLC7A11 inhibitor such that the brain cancer is treated in the cell. In some embodiments, the brain cancer comprises glioblastoma.

[0019] Additional features and advantages are described herein, and will be apparent from the following detailed description, drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The foregoing aspects and other features of the disclosure are explained in the following description, taken in connection with the accompanying drawings, wherein:

[0021] FIG. 1 shows ABL-dependent transcriptome of PC9 human EGFR mutant lung adenocarcinoma cells treated with ABL kinase specific allosteric inhibitor GNF5. Treatment occurred for 72 hours, and then RNA sequencing was performed to determine the ABL- dependent transcriptome. SLC7A11 is identified as the most statistically significant downregulated gene in n the transcriptome, as compared to PC9 control cells.

[0022] FIGS. 2A-2B show ABL2 kinase and SLC7A11 total RNA in samples of twenty (20) human tissues. FIG. 2A shows results of RNA sequencing of total ABL2 kinase RNA in each of the 20 tissues, showing that total RNA of ABL2 kinase is highest in the brain (whole), the cerebellum region of the brain, and in the fetal brain. FIG. 2B shows results of RNA sequencing of total SLC7A11 RNA in each of the same 20 tissues, showing that total RNA is similarly highest in the brain (whole), the cerebellum region of the brain, and in the fetal brain.

[0023] FIGS. 3A-B show correlation of expression of ABL2 and SLC7A11 with survival in lung adenocarcinoma patients. FIG. 3A shows that high expression of ABL2 results in decreased progression-free survival in patients with lung adenocarcinoma, as compared with low expression of ABL2 kinase. FIG. 3B similarly shows that high expression of SLC7A11 results in decreased progression-free survival in patients with lung adenocarcinoma, as compared with low expression of SLC7A11.

[0024] FIG. 4 shows western blot analysis of SLC7A11 expression across multiple cell lines including breast, glioma and lung cancer cells. All cell lines were plated at the same seeding density and harvested 72 hours later for western blot analysis. GAPDH levels in each cell line were also measured as a control. SLC7A11 protein expression levels were greater in lung and breast cancer cells.

[0025] FIGS. 5A-E show ABL kinase inhibition decreases SLC7A11 expression and impairs System xCT function in lung adenocarcinoma. FIG. 5A shows RT-PCR analysis of SLC7A11 mRNA in PC9 lung adenocarcinoma cells containing an ABL1/ABL2 shRNA mediated knockdown (shAA) as compared to the control cells (shSCR). SLC7A11 expression in shAA knockdown cells was reduced compared with the control cells. FIG. 5B shows RT- PCR analysis of SLC7A11 mRNA in PC9 lung adenocarcinoma cells treated with ABL- specific allosteric inhibitor GNF5 compared to PC9 cells treated with DMSO (control). SLC7A11 mRNA expression was reduced in cells treated with GNF5 as compared to the control cells. FIG. 5C shows protein analysis of SLC7A11 following ABL 1/2 knockdown (shAA) or SLC7A11 knockdown (shSLC7Al 1) via shRNAs. In both shAA and shSLC7Al 1 knockdown cells, SLC7A11 protein levels were reduced as compared to the control group. FIG. 5D shows functional analysis of intracellular glutathione, a surrogate marker for changes in SLC7A11 function. In both shAA and shSLC7Al 1 knockdown cells, the levels of intracellular glutathione are reduced as compared with the control. FIG. 5E shows functional analysis of intracellular glutamate within System xCT. In ABL2/ABL2 shRNA mediated knockdown (shAA), intracellular glutamate is increased. Two mutants containing SLC7A11 knockdown (shSLC7Al 1 #1 and #2) demonstrate varying increases in intracellular glutamate over the control vector sample.

[0026] FIGS. 6A-D provide additional support showing that ABL kinase inhibition decreases SLC7A11 expression and impairs system xCT function in lung adenocarcinoma. FIG. 6A shows SLC7A11 protein expression was reduced in Epidermal Growth Factor Receptor (“EGFR”) mutant lung adenocarcinoma cell lines PC9, H1975 and H1975 BrM3 following ABL kinase 1/2 shRNA mediated knockdown. As expected, ABL 1 and ABL2 expression were also reduced or eliminated in the ABL kinase 1/2 shRNA mediated knockdowns for each cell type. The same cell lines without ABL kinase 1/2 shRNA mediated knockdown (control) did not exhibit reduced expression of SLC7A11, ABL 1 or ABL 2. FIG. 6B shows that in PC9 lung cancer cells, SLC7A11 protein levels decrease only with ABL1 and ABL2 knockdown (shAA) FIG. 6C shows reduced extracellular glutamate in H1975 BrM3 cells with ABL 1/2 shRNA mediated knockdown as compared to the control. FIG. 6D shows treatment with ABL kinase specific inhibitor GNF5 decreases SLC7A11 protein expression in lung cancer xenografts. [0027] FIGS. 7A-B show SLC7A11 function inhibited by shRNA mediated ABL 1/2 knockdown is increased following treatment with H202, which enhances SLC7A11 function. Intracellular glutathione (FIG. 7A) and extracellular glutathione (FIG. 7B) are increased in both the PC9 control group and the PC9 shRNA mediated ABL 1/2 knockdown following treatment with H202, as compared to the untreated control and knockdown groups.

[0028] FIGS. 8A-B show conditioned media from ABL-deficient lung cancer cells fails to elicit tumor-induced neuronal cyclotoxicity. FIG. 8A shows dead rat cortical neurons, cultured 7 days in vitro (DIV7) and cultured for 18 hours in replacement media that was either fresh media, contained 5 mM glutamate, or tumor conditioned medium from PC9 cells containing ABL kinase 1/2 knockdown (shAA) or control shRNA (shSCR), or PC9 cells cultured with 200 mM sulphasalzine (SAS), an xCT inhibitor. After culture, neurons were stained using Live/Dead kit from Molecular Probes and counted using ImageJ software. N=2 biological replicates. FIG. 8B shows neurons cultured in conditioned media from PC9 shSCR cells or glutamate-conditioned medium showed fewer protrusions than neurons cultured in ABL-depleted PC9 shAA conditioned media, PC9 + SAS conditioned media, or fresh media. *p<0.05.

[0029] FIGS. 9A-B show ABL kinase inhibition decreases SLC7A11 expression in triple-negative breast cancer. FIG. 9A shows SLC7A11 protein levels are reduced following shRNA mediated knockdown of ABL kinase 1/2. FIG. 9B shows SLC7A11 protein levels are also reduced following pharmacologic inhibition of the ABL kinases with ABL allosteric inhibitors GNF5 or ABLOOl.

[0030] FIGS. 10A-C show ABL kinases modulate SLC7A11 expression in triplenegative breast cancer in a TAZ-dependent manner. FIG. 10A shows Western blot analysis of SUM 159 triple-negative breast cancer cells revealed SLC7A11 protein levels were rescued in shAA cells expressing constitutively activated TAZ4SA. FIG. 10B shows functional analysis of ABL-depleted SUM 159 cells showed glutathione levels were decreased to levels similar to SLC7A11 shRNA-expressing cells. FIG. IOC confirms the Western blot analysis as glutathione levels were also rescued in shAA cells expressing constitutively activated TAZ4SA. Glutathione levels were also at least partially restored in shSLC7Al l cells expressing constitutively active TAZ4SA.

[0031] FIGS. 11A-B show SLC7A11 function inhibited by shRNA mediated ABL 1/2 knockdown is marginally affected by H202 treatment. Intracellular glutathione (FIG. 11 A) is increased in MDA-MB-231 cells shRNA mediated ABL 1/2 knockdown following H202 treatment. However, intracellular glutathione is only slightly increased in SUM 159 shAA cells treated with H202, and extracellular glutathione (FIG. 11B) is similarly increased slightly in MDA231 shAA cells treated with H202, as compared to the untreated control and knockdown groups.

[0032] FIGS. 12A-D show ABL kinase inhibition decreases SLC7A11 expression and impairs System xCT function in gliomas. SLC7A11 protein levels are altered following shRNA mediated knockdown of ABL kinase 1/2 (FIG. 12A) or pharmacologic inhibition of the ABL kinases with the ABL allosteric inhibitor GNF5 (FIG. 12B) in glioma cells. Similarly, FIG. 12C shows S1C7A11 function is also impacted following ABL kinase 1/2 knockdown or pharmacologic inhibition. FIG. 12D shows functional analysis of ABL- depleted PC9 and U87 cells provide intracellular glutathione levels were decreased to levels similar to SLC7A11 shRNA-expressing cells.

[0033] FIGS. 13A-B show ABL inhibition impairs glioma cell survival. FIG. 13 A shows pharmacologic inhibition of ABL kinase inhibitor GNF5 decreases glioma cell survival. FIG. 13B shows U87 cell growth was decreased in sliAA cells.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0034] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

Definitions

[0035] Articles “a” and “an" are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0036] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.

[0037] The use herein of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as "including," "comprising,” or "having" certain elements are also contemplated as "consisting essentially of and "consisting of those certain elements. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alterative (“or”).

[0038] As used herein, the transitional phrase "consisting essentially of' (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP §2111.03. Thus, the term "consisting essentially of' as used herein should not be interpreted as equivalent to "comprising."

[0039] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0040] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0041] As used herein, "treatment,” “therapy" and/or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.

[0042] The term "effective amount" or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.

[0043] As used herein, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. The term "nonhuman animals" of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. In some embodiments, the subject comprises a human. In certain embodiments, the subject comprises a human having cancer or a CNS disorder.

[0044] As used herein, the term “central nervous system disorder” or “CNS disorder" refer to those CNS disorders characterized by the dysfunction on glutamate signaling. Examples include, but are not limited to, drug addiction, schizophrenia, amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, and the like.

[0045] "Administration" as it applies to a human, primate, mammal, mammalian subject, animal, veterinary subject, placebo subject, research subject, experimental subject, cell, tissue, organ, or biological fluid, refers without limitation to contact of an exogenous ligand, reagent, placebo, small molecule, pharmaceutical agent, therapeutic agent, diagnostic agent, or composition to the subject, cell, tissue, organ, or biological fluid, and the like. "Administration" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. "Administration" also encompasses in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell.

[0046] As used herein, “ABL kinase inhibitor” refers to any compound capable of disrupting, blocking, or inhibiting the expression and/or function (including the signal transduction pathway) of ABL kinase(s) (e.g., ABLl, ABL2), and/or any of its signal transduction components in a cell, including the Bcl-ABL pathway. The term “ABL kinase inhibitor” is meant to include one or more compounds capable of disrupting, blocking, or inhibiting the expression and/or function, i.e. the term may include two or more inhibitors that may be used in combination, including sequential or concomitant administration. The ABL kinase inhibitors as used with the present invention may be ABL kinase specific inhibitors. The ABL kinase inhibitors may be allosteric inhibitors.

[0047] As used herein, “SLC7A 11 inhibitor” refers to any compound capable of disrupting, blocking, or inhibiting the expression and/or function of SLC7A11 protein. The term “SLC7A11 inhibitor" is meant to include one or more compounds capable of disrupting, blocking, or inhibiting the expression and/or function, i.e. the term may include two or more inhibitors that may be used in combination, including sequential or concomitant administration.

[0048] As used herein, “an interfering oligonucleotide" refers to any oligonucleotide that interferes with, i.e. reduces, inhibits, or eliminates, the expression of an ABL kinase. Interfering oligonucleotides include aptamers and other oligonucleotide molecules as described herein.

[0049] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

ABL Kinases Regulates SLC7A11 Expression and Inhibition of ABL Kinases Decreases SLC7A11 mRNA Levels

Inhibition of ABL Kinases And/Or SLC7A11 Reduces Glutamate Export in Cells

[0050] The inventors have surprisingly discovered that the gene SLC7A11 is differentially regulated in Abelson (ABL) kinase-inhibited cells. In particular, SLC7AI1 mRNA levels are decreased following either ABL kinase genetic knockdown or pharmacological inhibition with an ABL kinase allosteric inhibitor. SLC7A11 is of importance as it has been shown to be overexpressed in multiple cancer types and regulates glutamate export from the cell.

Increased glutamate export can be detrimental to surrounding cells. In particular, neurons undergo glutamate excitotoxicity, a pathway of cell death more commonly associated with stroke and neurodegenerative disease. SLC7A11 expression has been associated with seizures, and elevated levels of SLC7A11 expression result in decreased survival and increased growth and expansion of glioma in subjects with malignant glioma. Robert SM, et al. (2015). However, regulation of SLC7A11 expression by ABL kinases is previously unknown.

[0051] Accordingly, one aspect of the present disclosure provides a method of downregulating glutamate export in a cell, the method comprising administering to a subject a therapeutically effective amount of an ABL kinase inhibitor and/or a SLC7A11 inhibitor such that the glutamate export is downregulated in the cell.

Inhibition of ABL Kinases And/Or SLC7A11 for the Treatment of CNS Disorders

[0052] Many CNS disorders are due to dysfunction in glutamate signaling. Glutamate is transported via excitatory amino acid transporters (“EAATs”) and system Xc- (also known as xCT) which transports cystine/glutamate. Impairment of either of these transporters results in a disruption in glutamate homeostasis and leads to a variety of CNS disorders.

[0053] Accordingly, a second aspect of the present disclosure provides a method of treating a CNS disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of an ABL kinase inhibitor and/or a SLC7A11 inhibitor such that the CNS disorder is treated in the subject. Inhibition of ABL Kinases And/Or SLC7A11 Reduces Neuron Cell Death

[0054] The release of glutamate by system xCT also leads to excitotoxicity, which is initiated by extrasynaptic N-methyl-D-aspartate (“NMDA”) receptors that result in neuronal death. Glutamate that is released from microglia has been shown to lead to oligodendrocyte death in culture and the rat optic nerve; however, an increase in system xCT activity also has been shown to have a protective effect by increasing the levels of glutathione.

[0055] Accordingly, a third aspect of the present disclosure provides a method of preventing and/or reducing neuron cell death in a subject, the method comprising administering to a subject a therapeutically effective amount of an ABL kinase inhibitor and/or a SLC7A11 inhibitor such that the neuron cell death in the subject is prevented and/or reduced.

Inhibition of ABL Kinases And/Or SLC7A11 for the Treatment of Cancer and CNS Disorders

[0056] The inventors’ discovery that inhibition of ABL kinases results in a downregulation of SLC7A11 indicates that both ABL kinases and SLC7A11 may serve as therapeutic targets in the treatment of cancer and CNS disorders. The ABL kinases, ABL1 and ABL2, are a family of non-receptor tyrosine kinases that regulate a wide variety of cellular processes during development and normal homeostasis, but can have deleterious effects on cell survival, proliferation, and cell-cell junction adhesion upon their upregulation following inflammation, tumorigenesis, and oxidative stress. Similarly, SLC7A11 has been shown to be overexpressed in multiple cancer types and regulates glutamate export from the cell. Glutamate excitotoxicity caused by upregulation of SLC7A11 has been shown to promote glioma growth. Furthermore, ABL2 and SLC7A1I expression are both highest in human brain tissue. See e.g., FIGS. 2A-B.

[0057] A fourth aspect of the present disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an ABL kinase inhibitor and/or a SLC7A11 inhibitor such that the cancer is treated in the subject. In some embodiments, the inhibitor is an ABL kinase specific inhibitor. In some embodiments, the inhibitor is a SLC7A11 inhibitor.

[0058] As is known in the art, a cancer is generally considered as uncontrolled cell growth. The methods of the present disclosure can be used to treat any cancer, and any metastases thereof, including, but not limited to, carcinoma, lymphoma, blastema, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer, endometrial carcinoma, salivary gland carcinoma, mesothelioma, kidney cancer, vulval cancer, pancreatic cancer, thyroid cancer, hepatic carcinoma, skin cancer, melanoma, brain cancer (e.g., gliomas, glioblastomas), neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma. In some embodiments, the cancer comprises a metastatic cancer. In some embodiments, the cancer comprises a brain cancer. In certain embodiments, the cancer comprises a glioblastoma.

[0059] In certain embodiments, the subject comprises a human having brain cancer, lung cancer or breast cancer. In some embodiments, the subject comprises a human having malignant glioma, non-small cell lung cancer or triple negative breast cancer. In certain embodiments, the subject comprises a human having drug addiction, schizophrenia, amyotrophic lateral sclerosis (“ALS”), Alzheimer’s disease, or Parkinson’s disease.

[0060] In certain embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

ABL Kinase Inhibitors

[0061] Exemplary ABL kinase inhibitors include, but are not limited to, GNF5 and ABL001. and combinations thereof and pharmaceutical compositions thereof. In certain embodiments, the ABL inhibitor is an allosteric inhibitor selected from the group consisting of GNF5, and ABL001. In some embodiments, the Abl kinase inhibitor comprises GNF5.

[0062] Additional Abl inhibitors that may be used with the compositions and methods disclosed herein are as follows: ABL (T315I) kinase Inhibitor AMBIT, ACTBI011, Adraine, AEG41174, Agacel, ALI8, Altanib, APG1351, APO-Imatinib, ARRY614, AT9283, AZD0424, BcrABL/Lyn Inhibitor, AB SCIENCE, Benznib, BL001, C-ABL Inhibitors HUM, Cadinib, Celonib, Dasanat, Dasatinib, ACCURE, Dasatinib HETERO, Dasatinib JODAS,

Dasatinib LIFEPHARMA FZE, Dasatinib SRS PHARMA, Dasatinib VALEANT, DCC2036, Defect Shoe, Degrasyns CALLISTO, Enliven, Fontrax, Gelike, Gistamel, Gleevec, Gleevec KEDEM, Gleevec-NP CAPSULUTION, GNF2, Glimatinib, Glinib, Glitive, GLYBULEN, HHGV678, HM95091, Hronileucem, HyNap-Nilo, I-Teenib, Iclusig, IkT-001, IkT-OOlPro, Imaget, Imakrebin, Imalek, Imanib, Imanix, Imarem, Imat Imatenil, Imatenil NEUTEC, Imatib, Imatinate, Imatinb, Imatinib ACCURE, Imatinib ADMAC, Imatinib ALLERGAN, Imatinib ALVOGEN, Imatinib AQVIDA, Imatinib ASCENDIS, Imatinib COOPER, Imatinib DENK, Imatinib Ecker hydrochloride, Imatinib ERIOCHEM, Imatinib, EUROFARMA, Imatinib FARMAPROJECTS, Imatinib FLAGSHIP BIOTECH, Imatinib GENEX, Imatinib INDIAN DRUGS, Imatinib JODAS, Imatinib LAFEDAR, Imatinib LIFEPHARMA FZE, Imatinib mesylate AMNEAL, Imatinib mesylate CAMUS, Imatinib mesylate CELOGEN, Imatinib mesylate CYGNUS, Imatinib mesylate CYNO PHARMACEUTICALS, Imatinib, mesylate DAIICHI SANKYO, Imatinib mesylate DOC, Imatinib mesylate EUROFARMA, Imatinib mesylate EXVASTAT, Imatinib mesylate HARVEST MOON, Imatinib mesylate HETERO, Imatinib mesylate LABORATORIOS INDUQUIMICA, Imatinib mesylate MEDAC, Imatinib Mesylate NAPROD, Imatinib mesylate NICHIIKO, Imatinib mesylate PHARMERJCA, Imatinib mesylate PRIME PHARMA, Imatinib mesylate SAVA, Imatinib mesylate SINO BIO, Imatinib Mesylate SRS PHARMA, Imatinib mesylate STERLING, Imatinib mesylate SYNTHON, Imatinib mesylate TAKATA, Imatinib mesylate ZENTIVA, Imatinib OSVAH Imatinib SALIUS, Imatinib UNITED BIOTECH, Imatinib VTVIMED, Imatinib WORLD MEDICINE, ImatiRel, Imatis, Imatoz, Imavec, Imavec HELM, Imicap, Imimark, Inivec INN0406, Itnib, Kimatinib, KW2449, Leukivec, Leutipol, Leuvec, Leuzek, Levin DR REDDYS, Liteda, LSI 04, Lupinib, MAAC002, Matinic, Meaxin, Megavec, Mesinib, Mitinab, Nibix, Nibix SOPHARMA, Nilotinib FARMAPROJECTS, Nilotinib JODAS, Nilotinib LIFEPHARMA FZE, NRCAN0I9, Nutab, ON012380, ON044580, ON 146040, PF114, PHA680626, Philachromin, Rembre, Sagitta, SAR103168, SGX393, SKLB1028, Sprycel, Sprytinib, Stimanib, Stritinib, SUN-K0706, SUN-K954, Supect, Tagonib, Tasigna, TG100598, Tibaldix, Timab, Tinima, Veenat NATCO, Veenat RADIANCE, Vek, VX680, Xin dimension, XL228, Zairanib, Ziatir, Zimitib and combinations thereof SLC7A11 Inhibitors [0063] Exemplaiy SLC7A11 inhibitors include, but are not limited to, PRO4051 (also known as Cpd X, CpdX, Pro 4051), SXC2023, and combinations thereof. In certain embodiments, the SLC7A11 inhibitor comprises PRO4051. In certain embodiments, the

SLC7A11 inhibitor comprises SXC2023.

[0064] Also contemplated by the present disclosure are other types of inhibitors of ABL kinases/Bcl-ABL pathways and/or SLC7A11 inhibitors, including but not limited to, the following: i. Aptamers

[0065] Aptamers, also called nucleic acid ligands, are nucleic acid molecules characterized by the ability to bind to a target molecule with high specificity and high affinity. Almost every aptamer identified to date is a non-naturally occurring molecule.

[0066] Aptamers to a given target (e.g. an ABL kinase(s)) or SLC7A11 may be identified and/or produced by the method of Systematic Evolution of Ligands by Exponential enrichment (SELEX™). Aptamers and SELEX are described in Tuerk and Gold (Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug. 3; 249(4968): 505- 10) and in W091/19813.

[0067] Aptamers may be DNA or RNA molecules and may be single stranded or double stranded. The aptamer may comprise chemically modified nucleic acids, for example in which the sugar and/or phosphate and/or base is chemically modified. Such modifications may improve the stability of the aptamer or make the aptamer more resistant to degradation and may include modification at the 2' position of ribose.

[0068] Aptamers may be synthesized by methods which are well known to the skilled person. For example, aptamers may be chemically synthesized, e.g. on a solid support.

[0069] Solid phase synthesis may use phosphoramidite chemistry. Briefly, a solid supported nucleotide is detritylated, then coupled with a suitably activated nucleoside phosphoramidite to form a phosphite triester linkage. Capping may then occur, followed by oxidation of the phosphite triester with an oxidant, typically iodine. The cycle may then be repeated to assemble the aptamer.

[0070] Aptamers can be thought of as the nucleic acid equivalent of monoclonal antibodies and often have Kd's in the nM or pM range, e.g. less than one of 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM. As with monoclonal antibodies, they may be useful in virtually any situation in which target binding is required, including use in therapeutic and diagnostic applications, in vitro or in vivo. In vitro diagnostic applications may include use in detecting the presence or absence of a target molecule.

[0071] Aptamers according to the present disclosure may be provided in purified or isolated form. Aptamers according to the present disclosure may be formulated as a pharmaceutical composition or medicament.

[0072] Suitable aptamers may optionally have a minimum length of one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. [0073] Suitable aptamers may optionally have a maximum length of one of 20, 21, 22,

23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides.

[0074] Suitable aptamers may optionally have a length of one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides. ii. Oligonucleotide Repression of ABL Kinase Expression or SLC7A11

Expression

[0075] Oligonucleotide molecules, particularly RNA, may be employed to regulate gene expression. These include antisense oligonucleotides, targeted degradation of mRNAs by small interfering RNAs (siRNAs), small molecules, post transcriptional gene silencing (PTGs), developmentally regulated sequence-specific translational repression of mRNA by micro-RNAs (miRNAs) and targeted transcriptional gene silencing.

[0076] An antisense oligonucleotide is an oligonucleotide, preferably single stranded, that targets and binds, by complementary sequence binding, to a target oligonucleotide, e.g. mRNA. Where the target oligonucleotide is an mRNA, binding of the antisense to the mRNA blocks translation of the mRNA and expression of the gene product. Antisense oligonucleotides may be designed to bind sense genomic nucleic acid and inhibit transcription of a target nucleotide sequence.

[0077] In view of the known nucleic acid sequences for ABL kinases and SLC7A11, oligonucleotides may be designed to repress or silence the expression of ABL kinases (e.g., those regulated by the ABL1 gene or ABL2 gene) or to repress or silence the expression of SLC7A11. Such oligonucleotides may have any length, but may preferably be short, e.g. less than 100 nucleotides, e.g. 10-40 nucleotides, or 20-50 nucleotides, and may comprise a nucleotide sequence having complete- or near-complementarity (e.g. 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity) to a sequence of nucleotides of corresponding length in the target oligonucleotide, e.g. the ABLl kinase mRNA, the ABL2 kinase mRNA, or the SLC7A11 mRNA. The complementary region of the nucleotide sequence may have any length, but is preferably at least 5, and optionally no more than 50, nucleotides long, e.g. one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

[0078] Repression of ABL kinase expression or SLC7A11 expression will preferably result in a decrease in the quantity of ABL kinase(s) and/or SLC7A11 expressed by a cell. For example, in a given cell the repression of ABL kinase by administration of a suitable nucleic acid will result in a decrease in the quantity of ABL kinase and/or SLC7A11 expressed by that cell relative to an untreated cell. Repression may be partial. Preferred degrees of repression are at least 50%, more preferably one of at least 60%, 70%, 80%, 85% or 90%. A level of repression between 90% and 100% is considered a 'silencing' of expression or function.

[0079] A role for the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci has been demonstrated. Double-stranded RNA (dsRNA)-dependent post transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific genes of homology for silencing in a short period of time. It acts as a signal to promote degradation of mRNA with sequence identity. A 20-nt siRNA is generally long enough to induce gene-specific silencing, but short enough to evade host response. The decrease in expression of targeted gene products can be extensive with 90% silencing induced by a few molecules of siRNA. RNAi based therapeutics have been progressed into Phase I, II and III clinical trials for a number of indications (Nature 2009 Jan. 22;

457(7228):426-433).

[0080] In the art, these RNA sequences are termed "short or small interfering RNAs" (siRNAs) or "microRNAs" (miRNAs) depending on their origin. Both types of sequence may be used to down-regulate gene expression by binding to complementary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein. siRNAs are derived by processing of long double stranded RNAs and when found in nature are typically of exogenous origin. Micro-interfering RNAs (miRNA) are endogenously encoded small non-coding RNAs, derived by processing of short hairpins. Both siRNA and miRNA can inhibit the translation of niRNAs bearing partially complimentary target sequences without RNA cleavage and degrade mRNAs bearing fully complementary sequences.

[0081] Accordingly, the present disclosure provides the use of oligonucleotide sequences for down-regulating the expression of ABL kinases and/or SLC7A11.

[0082] siRNA ligands are typically double stranded and, in order to optimize the effectiveness of RNA mediated down-regulation of the function of a target gene, it is preferred that the length of the siKNA molecule is chosen to ensure correct recognition of the siRNA by the RISC complex that mediates the recognition by the siRNA of the mRNA target and so that the siRNA is short enough to reduce a host response.

[0083] miRNA ligands are typically single stranded and have regions that are partially complementary enabling the ligands to form a hairpin. miRNAs are RNA genes which are transcribed from DNA but are not translated into protein. A DNA sequence that codes for a miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base pair to fomi a partially double stranded RNA segment The design of microRNA sequences is discussed in John et al, PLoS Biology, 11(2), 1862-1879, 2004.

[0084] Typically, the RNA ligands intended to mimic the effects of siRNA or miRNA have between 10 and 40 ribonucleotides (or synthetic analogues thereof), more preferably between 17 and 30 ribonucleotides, more preferably between 19 and 25 ribonucleotides and most preferably between 21 and 23 ribonucleotides. In some embodiments of the invention employing double-stranded siRNA, the molecule may have symmetric 3' overhangs, e.g. of one or two (ribo)nucleotides, typically a UU of dTdT 3' overhang. Based on the disclosure provided herein, the skilled person can readily design suitable siRNA and miRNA sequences, for example using resources such the Ambion siRNA finder. siRNA and miRNA sequences can be synthetically produced and added exogenously to cause gene downregulation or produced using expression systems (e.g. vectors). In a preferred embodiment the siRNA is synthesized synthetically.

[0085] Longer double stranded RNAs may be processed in the cell to produce siRNAs (see for example Myers (2003) Nature Biotechnology 21:324-328). The longer dsRNA molecule may have symmetric 3' or 5* overhangs, e.g. of one or two (ribo)nucleotides, or may have blunt ends. The longer dsRNA molecules may be 25 nucleotides or longer. Preferably, the longer dsRNA molecules are between 25 and 30 nucleotides long. More preferably, the longer dsRNA molecules are between 25 and 27 nucleotides long. Most preferably, the longer dsRNA molecules are 27 nucleotides in length. dsRNAs 30 nucleotides or more in length may be expressed using the vector pDECAP (Shinagawa et al., Genes and Dev., 17, 1340-5, 2003).

[0086] Another alternative is the expression of a short hairpin RNA molecule (shRNA) in the cell. shRNAs are more stable than synthetic siRNAs. A shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complimentary to the gene target. In the cell the shRNA is processed by DICER into a siRNA which degrades the target gene mRNA and suppresses expression. In a preferred embodiment the shRNA is produced endogenously (within a cell) by transcription from a vector. shRNAs may be produced within a cell by transfecting the cell with a vector encoding the shRNA sequence under control of a RNA polymerase III promoter such as the human HI or 7SK promoter or a RNA polymerase P promoter. Alternatively, the shRNA may be synthesised exogenously (in vitro) by transcription from a vector. The shRNA may then be introduced directly into the cell. Preferably, the shRNA molecule comprises a partial sequence of the ABL kinase or SLC7A11. Preferably, the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length. The stem of the hairpin is preferably between 19 and 30 base pairs in length. The stem may contain G-U pairings to stabilize the hairpin structure.

[0087] siRNA molecules, longer dsRNA molecules or miRNA molecules may be made recombinantly by transcription of a nucleic acid sequence, preferably contained within a vector. Preferably, the siRNA molecule, longer dsRNA molecule or miRNA molecule comprises a partial sequence of the ABL kinase or SLC7A11.

[0088] In one embodiment, the siRNA, longer dsRNA or miRNA is produced endogenously (within a cell) by transcription from a vector. The vector may be introduced into the cell in any of the ways known in the art. Optionally, expression of the RNA sequence can be regulated using a tissue specific (e.g. heart, liver, kidney or eye specific) promoter. In a further embodiment, the siRNA, longer dsRNA or miRNA is produced exogenously (in vitro) by transcription from a vector.

[0089] Suitable vectors may be oligonucleotide vectors configured to express the oligonucleotide agent capable of ABL kinase and/or SLC7A11 repression. Such vectors may be viral vectors or plasmid vectors. The therapeutic oligonucleotide may be incorporated in the genome of a viral vector and be operably linked to a regulatory sequence, e.g. promoter, which drives its expression. The term "operably linked" may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence are covalently linked in such a way as to place the expression of a nucleotide sequence under the influence or control of the regulatory sequence. Thus, a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of a nucleotide sequence which forms part or all of the selected nucleotide sequence.

[0090] Viral vectors encoding promoter-expressed siRNA sequences are known in the art and have the benefit of long-term expression of the therapeutic oligonucleotide. Examples include lentiviral (Nature 2009 Jan. 22; 457(7228):426-433), adenovirus (Shen et al., FEBS Lett 2003 Mar. 27; 539(1-3)111-4) and retroviruses (Barton and Medzhitov PNAS Nov. 12, 2002 vol. 99, no. 23 14943-14945).

[0091] In other embodiments a vector may be configured to assist delivery of the therapeutic oligonucleotide to the site at which repression of ABL kinase or SLC7A11 expression is required. Such vectors typically involve complexing the oligonucleotide with a positively charged vector (e.g., cationic cell penetrating peptides, cationic polymers and dendrimers, and cationic lipids); conjugating the oligonucleotide with small molecules (e.g., cholesterol, bile acids, and lipids), polymers, antibodies, and RNAs; or encapsulating the oligonucleotide in nanoparticulate formulations (Wang et al, AAPS J. 2010 December; 12(4): 492-503).

[0092] In one embodiment, a vector may comprise a nucleic acid sequence in both the sense and antisense orientation, such that when expressed as RNA the sense and antisense sections will associate to form a double stranded RNA.

[0093] Alternatively, siRNA molecules may be synthesized using standard solid or solution phase synthesis techniques which are known in the art. Linkages between nucleotides may be phosphodiester bonds or alternatives, for example, linking groups of the formula P(0)S, (thioate); P(S)S, (dithioate); P(0)NR'2; P(0)R'; P(0)OR6; CO; or CONR'2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through — O— or — S— .

[0094] Modified nucleotide bases can be used in addition to the naturally occurring bases, and may confer advantageous properties on siRNA molecules containing them.

[0095] For example, modified bases may increase the stability of the siRNA molecule, thereby reducing the amount required for silencing. The provision of modified bases may also provide siRNA molecules which are more, or less, stable than unmodified siRNA.

[0096] The term “modified nucleotide base” encompasses nucleotides with a covalently modified base and/or sugar. For example, modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5* position. Thus modified nucleotides may also include 2’ substituted sugars such as 2'-0-methyl-; 2-O-alkyl; 2'-0-allyl; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro-; 2-halo or azido-ribose, carbocyclic sugar analogues, a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose. [0097] Modified nucleotides are known in the art and include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles. These classes of pyrimidines and purines are known in the art and include pseudoisocytosine, N4,N4- ethanocytosine, 8-hydroxy-N6-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5 fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyl uracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1- methyladenine, 1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7- methylguanine, 5-methylaminomethyl uracil, 5-methoxy amino methyI-2-thiouracil, -D- mannosylqueosine, 5-methoxycarbonylmethyluracil, 5methoxyuracil, 2 methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid methyl ester, psueouracil, 2-thiocytosine, 5- methyl-2 thiouracil, 2-thiouracil, 4-thiouracil, 5methyluracil, N-uracil-5-oxyacetic acid methylester, uracil 5-oxyacetic acid, queosine, 2-thiocytosine, 5-propyIuracil, 5- propylcytosine, 5-ethyluracil, 5ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine, and 2,6,diaminopurine, methylpsuedouracil, 1-methylguanine, 1-methylcytosine.

[0098] Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and mammals are known in the art (Fire A, et al, 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485- 490 (2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119 (2001); Tuschl, T.

Chem. Biochem. 2, 239-245 (2001); Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000); Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15, 188-200 (2001); W00129058; W09932619, and Elbashir S M, et al., 2001 Nature 411:494-498).

[0099] Accordingly, the present disclosure provides a nucleic acid that is capable, when suitably introduced into or expressed within a mammalian, e.g. human, cell that otherwise expresses ABL kinase(s) or SLC7A11, of suppressing ABL kinase expression or SLC7A11 expression by RNAi.

[0100] The nucleic acid may have substantial sequence identity to a portion of the ABL kinase mRNA or SLC7A11 mRNA, or the complementary sequence to either said mRNA. [0101] The nucleic acid may be a double-stranded siRNA. (As the skilled person will appreciate, and as explained further below, a siRNA molecule may include a short 3' DNA sequence also.) [0102] Alteratively, the nucleic acid may be a DNA (usually double-stranded DNA) which, when transcribed in a mammalian cell, yields an RNA having two complementary portions joined via a spacer, such that the RNA takes the form of a hairpin when the complementary portions hybridize with each other. In a mammalian cell, the hairpin structure may be cleaved from the molecule by the enzyme DICER, to yield two distinct, but hybridized, RNA molecules.

[0103] Only single-stranded (i.e. non self-hybridized) regions of an mRNA transcript are expected to be suitable targets for RNAi. It is therefore proposed that other sequences very close in the ABL kinase mRNA transcript or SLC7A11 mRNA transcript, respectively, may also be suitable targets for RNAi.

[0104] Accordingly, the present disclosure provides nucleic acids that are capable, when suitably introduced into or expressed within a mammalian cell that otherwise expresses ABL kinase(s) or SLC7A11, of suppressing ABL kinase expression or SLC7A11 expression by RNAi, wherein the nucleic acid is generally targeted to the sequence of, or portion thereof, of the ABL kinase or SLC7A11, respectively.

[0105] By "generally targeted" the nucleic acid may target a sequence that overlaps with the ABL kinase or SLC7A11. In particular, the nucleic acid may target a sequence in the mRNA of human ABL kinase or human SLC7A11 that is slightly longer or shorter than one of ABL kinase or SLC7A11, but is otherwise identical to the respective native form.

[0106] It is expected that perfect identity/complementarity between the nucleic acid of the invention and the target sequence, although preferred, is not essential. Accordingly, the nucleic acid of the invention may include a single mismatch compared to the mRNA of the ABL kinase or SLC7A11. It is expected, however, that the presence of even a single mismatch is likely to lead to reduced efficiency, so the absence of mismatches is preferred. When present, 3* overhangs may be excluded from the consideration of the number of mismatches.

[0107] The term "complementarity" is not limited to conventional base pairing between nucleic acid consisting of naturally occurring ribo- and/or deoxyribonucleotides, but also includes base pairing between mRNA and nucleic acids of the invention that include nonnatural nucleotides.

[0108] In one embodiment, the nucleic acid (herein referred to as double-stranded siRNA) includes the double-stranded RNA sequences for the ABL kinase or SLC7A11, respectively. However, it is also expected that slightly shorter or longer sequences directed to the same region of the ABL kinase mRNA or the SLC7A11 mRNA will also be effective. In particular, it is expected that double-stranded sequences between 17 and 23 bp in length will also be effective.

[0109] The strands that form the double-stranded RNA may have short 3' dinucleotide overhangs, which may be DNA or RNA. The use of a 3' DNA overhang has no effect on siRNA activity compared to a 3' RNA overhang, but reduces the cost of chemical synthesis of the nucleic acid strands (Elbashir et al., 2001c). For this reason, DNA dinucleotides may be preferred.

[0110] When present, the dinucleotide overhangs may be symmetrical to each other, though this is not essential. Indeed, the 3^* overhang of the sense (upper) strand is irrelevant for RNAi activity, as it does not participate in mRNA recognition and degradation (Elbashir et al., 2001a, 2001b, 2001c).

[0111] While RNAi experiments in Drosophila show that antisense 3' overhangs may participate in mRNA recognition and targeting (Elbashir et al. 2001c), 3' overhangs do not appear to be necessary for RNAi activity of siRNA in mammalian cells. Incorrect annealing of 3’ overhangs is therefore thought to have little effect in mammalian cells (Elbashir et al.

2001c; Czauderna et al. 2003). [0112] Any dinucleotide overhang may therefore be used in the antisense strand of the siRNA. Nevertheless, the dinucleotide is preferably -UU or -UG (or -TT or -TG if the overhang is DNA), more preferably -UU (or -TT). The -UU (or -TT) dinucleotide overhang is most effective and is consistent with (i.e. capable of forming part of) the RNA polymerase III end of transcription signal (the terminator signal is TTTTT). Accordingly, this dinucleotide is most preferred. The dinucleotides AA, CC and GG may also be used, but are less effective and consequently less preferred.

[0113] Moreover, the 3' overhangs may be omitted entirely from the siRNA.

[0114] The present disclosure also provides single-stranded nucleic acids (herein referred to as single-stranded siRNAs) respectively consisting of a component strand of one of the aforementioned double-stranded nucleic acids, preferably with the 3'-overhangs, but optionally without. The present disclosure also provides kits containing pairs of such single- stranded nucleic acids, which are capable of hybridizing with each other in vitro to form the aforementioned double-stranded siRNAs, which may then be introduced into cells.

[0115] The present disclosure also provides DNA that, when transcribed in a mammalian cell, yields an RNA (herein also referred to as an shRNA) having two complementary portions which are capable of self-hybridizing to produce a double-stranded motif or a sequence that differs from any one of the aforementioned sequences by a single base pair substitution.

[0116] The complementary portions will generally be joined by a spacer, which has suitable length and sequence to allow the two complementary portions to hybridize with each other. The two complementary (i.e. sense and antisense) portions may be joined 5'-3' in either order. The spacer will typically be a short sequence, of approximately 4-12 nucleotides, preferably 4-9 nucleotides, more preferably 6-9 nucleotides.

[0117] Preferably the 5' end of the spacer (immediately 3' of the upstream complementary portion) consists of the nucleotides -UU- or -UG-, again preferably -UU- (though, again, the use of these particular dinucleotides is not essential). A suitable spacer, recommended for use in the pSuper system of OligoEngine (Seattle, Wash., USA) is UUCAAGAGA. In this and other cases, the ends of the spacer may hybridize with each other.

[0118] Similarly, the transcribed RNA preferably includes a 3' overhang from the downstream complementary portion. Again, this is preferably -UU or -UG, more preferably - UU.

[0119] Such shRNA molecules may then be cleaved in the mammalian cell by the enzyme DICER to yield a double-stranded siRNA as described above, in which one or each strand of the hybridized dsRNA includes a 3' overhang.

[0120] Techniques for the synthesis of the nucleic acids of the invention are of course well known in the art.

[0121] The skilled person is well able to construct suitable transcription vectors for the DNA of the present disclosure using well-known techniques and commercially available materials. In particular, the DNA will be associated with control sequences, including a promoter and a transcription termination sequence.

[0122] Of particular suitability are the commercially available pSuper and pSuperior systems of OligoEngine (Seattle, Wash., USA). These use a polymerase-III promoter (HI) and a T₅ transcription terminator sequence that contributes two U residues at the 3' end of the transcript (which, after DICER processing, provide a 3' UU overhang of one strand of the siRNA).

[0123] Another suitable system is described in Shin et al. (RNA, 2009 May; 15(5): 898- 910), which uses another polymerase-III promoter (U6).

[0124] The double-stranded siRNAs of the present disclosure may be introduced into mammalian cells in vitro or in vivo using known techniques, as described below, to suppress expression of the ABL kinase and/or expression of SLC7A11. [0125] Similarly, transcription vectors containing the DNAs of the present disclosure may be introduced into cells (e.g., cancer cells) in vitro or in vivo using known techniques, as described below, for transient or stable expression of RNA, again to suppress expression of the ABL kinase and/or expression of SLC7A11.

[0126] Accordingly, the present disclosure also provides a method of suppressing ABL kinase expression and/or expression of SLC7A11 in a mammalian, e.g. human, cell, the method comprising administering to the cell a double-stranded siRNA of the present disclosure or a transcription vector of the present disclosure.

[0127] The present disclosure further provides the double-stranded siRNAs of the present disclosure and the transcription vectors of the present disclosure, for use in a method of treatment, preferably a method of treating a cancer or CNS disorder in a subject.

[0128] The present disclosure further provides the use of the double-stranded siRNAs of the present disclosure and the transcription vectors of the present disclosure in the preparation of a medicament for the treatment of cancer or a CNS disorder in a subject.

[0129] The present disclosure further provides a composition comprising a double- stranded siRNA of the present disclosure or a transcription vector of the present disclosure in admixture with one or more pharmaceutically acceptable carriers. Suitable carriers include lipophilic carriers or vesicles, which may assist in penetration of the cell membrane.

[0130] Materials and methods suitable for the administration of siRNA duplexes and DNA vectors of the present disclosure are well known in the art and improved methods are under development, given the potential of RNAi technology.

[0131] Generally, many techniques are available for introducing nucleic acids into mammalian cells. The choice of technique will depend on whether the nucleic acid is transferred into cultured cells in vitro or in vivo in the cells of a patient. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE dextran and calcium phosphate precipitation. In vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al. (2003)

Trends in Biotechnology 11, 205-210). [0132] In particular, suitable techniques for cellular administration of the nucleic acids of the present disclosure both in vitro and in vivo are disclosed in the following articles:

[0133] General reviews: Borkhardt, A. 2002. Blocking oncogenes in malignant cells by RNA interference-new hope for a highly specific cancer treatment? Cancer Cell. 2:167-8. Hannon, G. J. 2002. RNA interference. Nature. 418:244-51. McManus, M. T., and P. A. Sharp. 2002. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet. 3:737- 47. Scherr, M, M. A. Morgan, and M. Eder. 2003b. Gene silencing mediated by small interfering RNAs in mammalian cells. Curr Med Chem. 10:245-56. Shuey, D. J., D. E. McCallus, and T. Giordano. 2002. RNAi: gene-silencing in therapeutic intervention. Drug

Discov Today. 7:1040-6. [0134] Systemic delivery using liposomes: Lewis, D. L., J. E. Hagstrom, A. G. Loomis, J. A. Wolff, and H. Herweijer. 2002. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat Genet. 32:107-8. Paul, C. P., P. D. Good, I. Winer, and D. R. Engelke. 2002. Effective expression of small interfering RNA in human cells. Nat Biotechnol. 20:505-8. Song, E., S. K. Lee, J. Wang, N. Ince, N. Ouyang, J. Min, J. Chen, P. Shankar, and J. Lieberman. 2003. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med. 9:347-51. Sorensen, D. R., M. Leirdal, and M. Sioud. 2003. Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J Mol Biol. 327:761-

6.

[0135] Virus mediated transfer: Abbas-Terki, T., W. Blanco-Bose, N. Deglon, W. Pralong, and P. Aebischer. 2002. Lentiviral-mediated RNA interference. Hum Gene Ther. 13:2197-201. Barton, G. M., and R. Medzhitov. 2002. Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci USA. 99:14943-5. Devroe, E., and P. A. Silver. 2002. Retrovirus-delivered siRNA. BMC Biotechnol. 2:15. Lori, F., P. Guallini, L. Galluzzi, and J. Lisziewicz. 2002. Gene therapy approaches to HIV infection. Am J Pharmacogenomics. 2:245-52. Matta, H., B. Hozayev, R. Tomar, P. Chugh, and P. M. Chaudhary. 2003. Use of lentiviral vectors for delivery of small interfering RNA. Cancer Biol Ther. 2:206-10. Qin, X. F., D. S. An, I. S. Chen, and D. Baltimore. 2003. Inhibiting HTV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci USA. 100:183-8. Scherr, M., K. Battmer, A. Ganser, and M. Eder. 2003a. Modulation of gene expression by lentiviral-mediated delivery of small interfering RNA. Cell Cycle. 2:251-7. Shen, C., A. K. Buck, X. Liu, M. Winkler, and S. N. Reske. 2003. Gene silencing by adenovirus-delivered siRNA. FEBS Lett. 539: 111-4.

[0136] Peptide delivery: Morris, M. C., L. Chaloin, F. Heitz, and G. Divita. 2000. Translocating peptides and proteins and their use for gene delivery. Curr Opin Biotechnol. 11:461-6. Simeoni, F., M. C. Morris, F. Heitz, and G. Divita. 2003. Insight into the mechanism of the peptide-based gene delivery system MPG: implications for deliveiy of siRNA into mammalian cells. Nucleic Acids Res. 31:2717-24. Other technologies that may be suitable for delivery of siRNA to the target cells are based on nanoparticles or nanocapsules such as those described in U.S. Pat. Nos. 6,649, 192B and 5,843,509B.

Administration of ABL Kinase And/Or SLC7A11 Inhibitors

[0137] One or more ABL kinase inhibitors, one or more SLC7A11 inhibitors, or both may be administered to a subject, either alone or as a composition comprising the ABL kinase inhibitor and/or the SLC7A11 inhibitor and a pharmaceutically acceptable carrier/excipient (i.e., a pharmaceutical composition), in an amount sufficient to induce an appropriate response in the subject.

[0138] An "effective amount" as used herein means an amount which provides a therapeutic or prophylactic benefit. Effective amounts of the compositions/pharmaceutical compositions provided herein can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0139] An effective amount of the composition(s) described herein may be given in one dose, but is not restricted to one dose. Thus, the administration can be two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more, administrations of the composition(s) disclosed herein. Where there is more than one administration in the present methods, the administrations can be spaced by time intervals of one minute, two minutes, three, four, five, six, seven, eight, nine, ten, or more minutes, by intervals of about one hour, two hours, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and so on. In the context of hours, the term "about" means plus or minus any time interval within 30 minutes. The administrations can also be spaced by time intervals of one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinations thereof. The invention is not limited to dosing intervals that are spaced equally in time, but encompass doses at non-equal intervals, such as a priming schedule consisting of administration at 1 day, 4 days, 7 days, and 25 days, just to provide a non-limiting example.

[0140] A "pharmaceutically acceptable excipient and/or carrier" or "diagnostically acceptable excipient and/or carrier" includes but is not limited to, sterile distilled water, saline, phosphate buffered solutions, amino acid-based buffers, or bicarbonate buffered solutions. An excipient selected and the amount of excipient used will depend upon the mode of administration. Administration comprises an injection, infusion, or a combination thereof. Any suitable combination of pharmaceutically acceptable carriers or excipients may be used, and as used herein the phrase “carrier or excipient" is meant to be inclusive of any individual carrier or excipient, or any combination of carrier(s) and/or excipient(s).

[0141] An effective amount for a particular subject/patient may vary depending on factors such as the condition being treated, the overall health of the patient, the route and dose of administration and the severity of side effects. Guidance for methods of treatment and diagnosis is available (see, e.g., Maynard, et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

[0142] A dosing schedule of, for example, once/week, twice/week, three times/week, four times/week, five times/week, six times/week, seven times/week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, and the like, is available for the invention. The dosing schedules encompass dosing for a total period of time of, for example, one week, two weeks, three weeks, four weeks, five weeks, six weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, and twelve months.

[0143] Provided are cycles of the above dosing schedules. The cycle can be repeated about, e.g., every seven days; every 14 days; every 21 days; every 28 days; every 35 days; 42 days; every 49 days; every 56 days; every 63 days; every 70 days; and the like. An interval of non-dosing can occur between a cycle, where the interval can be about, e.g., seven days; 14 days; 21 days; 28 days; 35 days; 42 days; 49 days; 56 days; 63 days; 70 days; and the like. In this context, the term "about" means plus or minus one day, plus or minus two days, plus or minus three days, plus or minus four days, plus or minus five days, plus or minus six days, or plus or minus seven days.

[0144] The composition(s) according to the present disclosure may also be administered with one or more additional therapeutic agents. Methods for co-administration with an additional therapeutic agent are well known in the art (Hardman, et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). [0145] Co-administration need to refer to administration at the same time in an individual, but rather may include administrations that are spaced by hours or even days, weeks, or longer, as long as the administration of the one or more therapeutic agents is the result of a single treatment plan. The co-administration may comprise administering the composition(s) of the present disclosure before, after, or at the same time as the additional therapeutic agent. By way of example, the composition(s) of the present disclosure may be given as an initial dose in a multi-day protocol, with additional therapeutic agent(s) given on later administration days; or the additional therapeutic agent(s) given as an initial dose in a multi-day protocol, with the composition^) of the present disclosure given on later administration days. On another hand, one or more additional therapeutic agent(s) and the composition(s) of the present disclosure may be administered on alternate days in a multi-day protocol. In still another example, a mixture of one or more additional therapeutic agent(s) and the compositions of the present disclosure may be administered concurrently. This is not meant to be a limiting list of possible administration protocols.

[0146] An effective amount of a therapeutic agent is one that will decrease or ameliorate the symptoms normally by at least 10%, more normally by at least 20%, most normally by at least 30%, typically by at least 40%, more typically by at least 50%, most typically by at least 60%, often by at least 70%, more often by at least 80%, and most often by at least 90%, conventionally by at least 95%, more conventionally by at least 99%, and most conventionally by at least 99.9%.

[0147] Formulations of the one or more therapeutic agents may be prepared for storage by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

[0148] The following Examples are provided by way of illustration and not by way of limitation. EXAMPLES

Materials and Methods

[0149] Cell culture. Rat cortical neurons were panned to remove astrocytes and cultured for seven (7) days in vitro (DIV7) to allow for neurons to polarize. On DIV7, half of the media was removed and replaced with either fresh media, 5 mM glutamate, or tumor conditioned medium from PC9 cells harboring shRNAs targeting ABL1/ABL2 (shAA) or control shRNA (shSCR), or PC9 cells were cultured with 200 mM sulphasalzine (SAS), an xCT inhibitor. PC9 cells were cultured in matching neurobasal media. Neurons were cultured for 18 hours in tumor conditioned medium and then stained using the Live/Dead kit from Molecular Probes, and dead cells were counted using ImageJ software. N=2 biological replicates.

[0150] Cell Lines: Breast cancer cell lines MDA-231 (MDA-MB-231), MDA-231-BrM, MDA-231-BRM2a and SUM- 159 were triple negative (ER-, PR-, Her2-), lacking expression of estrogen receptors (ER), progesterone receptors (PR) and amplification of human epidermal growth factor receptor 2 (Her2). Lung cancer cell lines PC-9, H460, HCC827, H358, H1975 and H1975-BrM were epidermal growth factor receptor (EGFR) mutant. Glioma cell lines U-87, U-138, CT-2A and LN-18 have a range of mutations, but each lacked driver oncogenes. Additional features: MDA-231-BrM, MDA-231-BRM2a, and H1975-BrM were brain metastatic. All cell lines were cultured at 37°C and 5% CO₂, as follows:

- MDA-231, MDA-231 -BrM and CT-2A were cultured in DMEM + 10% FBS;

- SUM-159 was cultured in F-12, 5% FBS, 1% HEPES, 0.125% insulin, 0.004% hydrocortisone;

- U-87 and U-138 were cultured in MEM + 10% FBS;

- Lung cancer cell lines were cultured in RPMI, 10% FBS, 1% HEPES, 1% NaPy, 0.5% glucose.

[0151] Real-time RT-oPCR. RNA was isolated from cells using an RNA isolation kit (GE-25050071), and complementary DNA was synthesized using oligo(dT) primers. Realtime PCR was performed using iQ SYBR Green Supermix (BioRad-1708882). The primers used were as follows:

Table 1: List of RT-qPCR Primers

[0152] Analysis was performed using a BioRad CFX384 real-time machine and CFX Manager software. PCR assays were performed in duplicate.

[0153] Mice. hairless; nu; Whn-, were purchased from

the Jackson Laboratory. PC9 lung cancer cells were administered and once tumors had reached 100-200 mm³, mice were either as administered ABL kinase inhibitor GNF5 two times each day for 1 week, while the other group was administered DMSO The mice were monitored daily for tumor growth, weight loss and signs of overall distress and euthanized in an C02 chamber at the completion of testing. All experiments were performed under the Duke University IACUC approved protocols: A098- 16-04 and A130-16-06. Mice were evaluated for differential response to drug (ABL kinase inhibitors or DMSO control) and inhibition of SLC7A11.

[0154] Inhibitors. GNF5 (N-(2-Hydroxyethyl)-3-(6-(4-(trifIuoromethoxy)phenylamino) pyrimidin-4-yI)benzamide) and ABL001 were synthesized at the Duke University’s Small Molecule Synthesis Facility, and validated by LC-MS and lH-NMR/FT-IR spectra and with cell-based assays that confirm potencies and cell signaling inhibitory activities. Sulfasalazine (SAS) was purchased commercially (Sigma Aldrich). For in vivo experiments, GNF5 was prepared in a suspension with 0.5% methylcellulose and 0.5% Tween-80 at a concentration of 10mg/mL, and mice were treated with SOmg/kg b.i.d. via oral gavage.

[0155] Statistical Analysis. Mouse experiments: Mouse numbers per group will be determined through statistical power calculations (a=0.05); 10 mice per group allows for 90% power to detect inter-group differences of 50% assuming intra-group variability of 25%. For Kaplan-Meier survival analysis, p values will be calculated using log-rank (Mantel-Cox) testing. Statistical comparisons of 2 groups will be conducted using Student’s i tests (unpaired, two-tailed). For comparisons involving more than two groups, data will be evaluated by ANOVA followed by Fisher post-hoc testing; p<0.05 is statistically significant. In vitro experiments: Each experiment will be performed at least 3 times with triplicate samples. Statistical comparison between control and experimental groups will be analyzed by ANOVA with post-hoc t-test or Tukey’s test. To increase statistical power of treatment groups, different experimental groups will be compared together in each assay via multi way- ANOVA.

Example 1: ABL kinases regulate SLC7A11 expression and System xCT function.

[0156] To identify potential genes relevant to the ABL-dependent transcriptome, PC9 human EGFR mutant lung adenocarcinoma cells were treated with ABL kinase allosteric inhibitor GNF5 for 72 hours. RNA sequencing was performed to determine the ABL- dependent transcriptome. SLC7A11 was identified as the most statistically significant downregulated gene. FIG. 1.

[0157] Several tests were performed to determine whether SLC7A11 is regulated by ABL kinases. Real-time polymerase chain reaction (“RT-PCR”) verified a reduction in SLC7A11 mRNA levels following ABL kinase pharmacologic inhibition or genetic knockdown. See FIGS. 5A-B Specifically, FIG. 5A shows a significant reduction in SLC7A11 mRNA levels when PC9 lung adenocarcinoma cells included a shRNA mediated double knockdown of ABL1 and ABL2 (shAA) (N=4, ***P < 0.0005). Furthermore, FIG. 5B shows a significant reduction in SLC7A11 mRNA in PC9 lung adenocarcinoma cells treated with ABL-specific allosteric inhibitor GNF5. (N=2, **P < 0.005). Western blotting also revealed a significant reduction in SLC7A11 protein levels for shRNA mediated double knockdown of ABL1 and ABL2 (shAA) and for shRNA mediated knockdown of SLC7A11 (shSLC7All). FIG. 5C. Finally, both shAA and ShSLC7Al 1 knockdowns show a statistically significant change in intracellular glutathione, which is a surrogate marker for changes in SLC7A11 function. In this case, shAA (***P < 0.0005) and shSLC7All (**P < 0.005) knockouts demonstrate a marked change in function of SLC7A11. FIG. 5D. Conversely, shAA knockouts resulted in increased levels of intracellular glutamate. FIG. 5E.

[0158] A glutamate functional test showed ABL kinase inhibition caused a similar effect on glutamate export as SLC7A11 inhibition. SLC7A11 has been shown to be transcriptionally regulated by NRF2 and previous studies in the lab have shown that ABL kinases enable NRF2 to enter the nucleus and promote transcription of target genes. Thus, this regulatory mechanism could explain ABL kinase control of SLC7A11 expression. Example 2: Effect of ABL kinase inhibition on SLC7A11 expression and function

[0159] To determine the effects of ABL kinase regulation of SLC7A11 and its effect on SLC7A11 expression and function, experiments were performed to identify the ability of ABL kinases to modulate system xCT function through decreased SLC7A11 expression in lung cancer, as initially predicted based on the tests performed in the Example above, and FIGS. 5A-E.

Administration of ABL kinase inhibitors and/or SLC7A11 inhibitors to treat lung cancer [0160] The tests performed confirmed that double knockout of ABL kinase 1 and ABL kinase 2 (shAA) inhibits ABLl, ABL2 and SLC7A11 expression in PC9 EGFR mutant lung adenocarcinoma cells, and inhibits ABLl and ABL2 expression in H1975 and H1975 BrM3 cells, with reduced expression of SLC7A11 in both cancer lines. See. FIG. 6A. Further tests were performed to confirm that shRNA mediated double knockdown of ABLl and ABL2 (shAA) is inhibits expression of ABLl, ABL2 and SLC7A11 in PC9 cells. FIG. 6B. To test the effect of ABL kinase inhibitor GNF5, mice were administered PC9 lung cancer cells and once tumors had reached 100-200 mm³, the mice were placed into treatment groups based on the tumor size, and one group was administered ABL kinase inhibitor GNF5 two times each day for 1 week, while the other group was administered DMSO. FIG. 6D. The results show a statistically significant reduction in size and weight of the tumors in the group that received treatment with GNF5. Id. Moreover, SLC7A11 expression was also inhibited in the group that received treatment with GNF5. Id.

Administration of ABL kinase inhibitors and/or SLC7A11 inhibitors to treat triple-negative breast cancer

[0161] Additional testing in other cell lines demonstrate that shRNA mediated double knockdown of ABLl and ABL2 (shAA) is inhibits expression of ABL1, ABL2 and SLC7A11 in triple-negative breast cancer cell lines MDA231, MDA231 BrM2a and SUM 159. FIG. 9B. Administration of GNF5 reduced expression of SLC7A11 in triplenegative breast cancer cell lines MDA231 and SUM 159, and administration of ABL001 also reduced expression of SLC7A11 in cancer cell line SUM 159. FIG. 9B. Further studies in triple-negative breast cancer provide evidence that ABL kinases modulate SLC7A11 expression in a TAZ-dependent manner. Specifically, in both shRNA mediated double knockout of ABLl and ABL2 (shAA) and knockout of SLC7A11 (shSLC7Al 1), intracellular glutathione is significantly reduced to levels similar to SLC7A11 shRNA-expressing cells in SUM159 cells, as compared to the control. FIG. 10A and 10B. However, when shAA cells express constitutively activated TAZ4SA, SLC7A11 protein levels were restored to control levels. FIG. 10C.

Administration of ABL kinase inhibitors and/or SLC7A 11 inhibitors to treat glioma

[0162] In addition to the testing performed on glioma cells in Example 1, additional testing shows that ABL kinase inhibition impairs glioma cell survival. FIGS. 13A-B. Specifically, the testing demonstrated that pharmacologic inhibition of ABL kinase inhibitor GNF5 decreases glioma cell survival. FIG. 13A. Further, glioma U87 cell growth was decreased in shAA cells. FIG. 13 B.

Discussion

[0163] As described above, the present disclosure provides support for use of ABL kinase inhibitors and/or SLC7A11 inhibitors in the treatment of several types of cancer, including but not limited to solid tumors, gliomas and other brain cancer, lung cancer and metastases of the same. New therapies and treatments are needed for cancer, and CNS disorders. Lung cancer often metastasizes before a patient is diagnosed, and significantly, metastasis is often to the brain. Effective treatments for lung cancer, whether it has metastasized at die time of diagnosis, are important for managing and even reducing the cancer.

[0164] Furthermore, as it is known that that both SLC7A11 and ABL2 mRNA expression are highest in the brain (FIGS. 2A-B), it was hypothesized that inhibition of ABL2 would cross the blood brain barrier, providing neuroprotective effects by disrupting brain metastases-mediated neurotoxicity, by inhibiting the function of the SLC7A11/xCT cystine- glutamate transporter expressed in the brain metastatic cells, as well as gliomas. As described above, inhibition of ABL kinases downregulates SLC7A11, including across the blood brain barrier, providing a neuroprotective effect to a patient. Perhaps more importantly, the findings described in the Examples above of have a profound impact in the treatment of these patients. There are currently no durable treatment options for lung cancer patients suffering from brain metastases.

[0165] Given the findings described in the Examples showing that ABL kinase inhibition decreases SLC7A11 expression and impairs system xCT functions as well as intracellular and extracellular glutamate, it was investigated whether ABL kinase inhibition and/or SLC7A11 inhibition also impairs tumor growth and metastasis by inhibiting GSH production in cancer cells through the same downregulation. [0166] The findings described herein support that inhibition of the ABL kinases promotes cells treated with one or more or more ABL kinase inhibitors, one or more SLC7A11 inhibitors, or both, including GNF5, ABL001, PRO4051 and SXC2023. [0167] In summary, ABL kinase inhibition can be a therapeutic strategy for cancers, including solid tumors, metastatic cancers and gliomas, as well as triple-negative breast cancer and CNS disorders. ABL kinase inhibition is fundamentally associated with epithelial cell differentiation, which is modulated in many disease states. The unexpected finding that ABL kinases regulate SLC7A11 expression expand therapeutic uses for both ABL kinase inhibitors and SLC7A11 inhibitors to include neuroprotective applications and downregulation of glutamate export in cells, among others.

[0168] Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the disclosure pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0169] One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present disclosure is presently representative of embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the disclosure as defined by the scope of the claims.

Claims

We claim:

1. A method of downregulating glutamate export in a cell, the method comprising administering to the subject a therapeutically effective amount of one or more ABL kinase inhibitors and/or SLC7A11 inhibitors.

2. A method of treating a central nervous system (CNS) disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more ABL kinase inhibitors and/or SLC7A11 inhibitors.

3. A method of preventing and/or reducing neuron cell death in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more ABL kinase inhibitors and/or SLC7A11 inhibitors.

4. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of one or more ABL kinase inhibitors and/or SLC7A11 inhibitors.

5. The method of claim 4, wherein the cancer comprises a metastatic cancer.

6. The method of claim 4, wherein the cancer is selected from the group consisting of lung cancer, brain cancer and breast cancer.

7. The method of any one of claims 4-6, wherein the breast cancer is triple-negative breast cancer.

8. The method of any one of claims 4-6, wherein the brain cancer comprises glioblastoma.

9. The method of any one of claims 1-8, wherein the one or more ABL kinase inhibitors comprises an allosteric inhibitor.

10. The method of any one of claims 1 -9, wherein the one or moe ABL kinase inhibitors is GNF5 or ABL001 , or combinations thereof.

11. The method of any one of claims 1-10, wherein the one or more SLC7A11 inhibitor comprises PRO4051 or SXC2023, or combinations thereof.