US 2016O138113A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0138113 A1 SEVENICH et al. (43) Pub. Date: May 19, 2016
(54) METHODS AND COMPOSITIONS FOR THE Related U.S. Application Data DLAGNOSIS, PROGNOSIS AND TREATMENT (60) Provisional application No. 61/836,993, filed on Jun. OF BRAIN METASTASIS 19, 2013. (71) Applicant: MEMORIAL SLOAN-KETTERING Publication Classification CANCER CENTER, New York, NY (US) (51) Int. Cl. CI2O I/68 (2006.01) (72) Inventors: Lisa Katharina Hildegard (52) U.S. Cl. SEVENICH, New York, NY (US); CPC ...... CI2O I/6886 (2013.01); C12O 2600/158 Johanna Alexandra JOYCE, New York, (2013.01); C12O 2600/16 (2013.01); C12O NY (US) 2600/1 18 (2013.01); C12O 2600/106 (2013.01) (21) Appl. No.: 14/899.720 (57) ABSTRACT The present disclosure describes a method for determining (22) PCT Filed: Jun. 19, 2014 metastasis-free survival of a subject with cancer based on the differential expression of genes that are associated with (86). PCT No.: PCT/US2O14/043291 metastasis of the cancer to the brain, bone and/or lung. Detec S371 (c)(1), tion of the expression level of these genes in a sample from the
(2) Date: Dec.e JLV918, 2015 Subject can identify an individual who is at risk for metastasis. Patent Application Publication May 19, 2016 Sheet 1 of 77 US 2016/0138113 A1
£-3-3 isix Orgar-specific is -is: x: waii:its {K}{{{{{}}rised 388tastasis 8 fety s:
FIGURE 1A Patent Application Publication May 19, 2016 Sheet 2 of 77 US 2016/0138113 A1
First2. Corport : 123First. Corportent3.375
FIGURE 1B Patent Application Publication May 19, 2016 Sheet 3 of 77 US 2016/0138113 A1
FIGURE 1C Patent Application Publication May 19, 2016 Sheet 4 of 77 US 2016/0138113 A1
FIGURE 1D Patent Application Publication May 19, 2016 Sheet 5 of 77 US 2016/0138113 A1
Brain Cross-species scatter plot
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FIGURE 2A Patent Application Publication May 19, 2016 Sheet 6 of 77 US 2016/0138113 A1
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FIGURE 2B Patent Application Publication May 19, 2016 Sheet 7 of 77 US 2016/0138113 A1
Brain BOne 100 100 80 80 als 60 60 GD 40 SD 40 h : 8 20 is 20 8 i o i O S s O 20 40 60 80 100 O 20 40 60 80 100 Time (months) Time (months)
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FIGURE 2C Patent Application Publication May 19, 2016 Sheet 8 of 77 US 2016/0138113 A1
FIGURE 2D Patent Application Publication May 19, 2016 Sheet 9 of 77 US 2016/0138113 A1
2 3 4 5 6 Patie: 2 3 4 5, 6
FIGURE 2E
Primary breast carcer Brain retastases
CD68+ macrophages
FIGURE 2F Patent Application Publication May 19, 2016 Sheet 10 of 77 US 2016/0138113 A1
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FIGURE 3A Patent Application Publication May 19, 2016 Sheet 11 of 77 US 2016/0138113 A1
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FIGURE 3B Patent Application Publication May 19, 2016 Sheet 12 of 77 US 2016/0138113 A1
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FIGURE 3C Patent Application Publication May 19, 2016 Sheet 13 of 77 US 2016/0138113 A1
FIGURE 3D Patent Application Publication May 19, 2016 Sheet 14 of 77 US 2016/0138113 A1
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FIGURE 3E Patent Application Publication May 19, 2016 Sheet 15 of 77 US 2016/0138113 A1
Patent Application Publication May 19, 2016 Sheet 16 of 77 US 2016/0138113 A1
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FIGURE 4B Patent Application Publication May 19, 2016 Sheet 17 of 77 US 2016/0138113 A1
24h time point
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FIGURE 5A Patent Application Publication May 19, 2016 Sheet 18 of 77 US 2016/0138113 A1
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FIGURE 5B Patent Application Publication May 19, 2016 Sheet 19 of 77 US 2016/0138113 A1
xtravascular Extravasating Extravascular 24h time point 48h time point
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FIGURE 5C Patent Application Publication May 19, 2016 Sheet 20 of 77 US 2016/0138113 A1
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FIGURE 5D Patent Application Publication May 19, 2016 Sheet 21 of 77 US 2016/0138113 A1
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FIGURE 6E Patent Application Publication May 19, 2016 Sheet 26 of 77 US 2016/0138113 A1
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FIGURE 11A Patent Application Publication May 19, 2016 Sheet 53 of 77 US 2016/0138113 A1
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FIGURE 11 B Patent Application Publication May 19, 2016 Sheet 54 of 77 US 2016/0138113 A1
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FIGURE 11 C Patent Application Publication May 19, 2016 Sheet 55 of 77 US 2016/0138113 A1
FIGURE 11 D Patent Application Publication May 19, 2016 Sheet 56 of 77 US 2016/0138113 A1
e lung
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FIGURE 11E Patent Application Publication May 19, 2016 Sheet 57 of 77 US 2016/0138113 A1
FIGURE 11 F Patent Application Publication May 19, 2016 Sheet 58 of 77 US 2016/0138113 A1
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Figure 12B Patent Application Publication May 19, 2016 Sheet 60 of 77 US 2016/0138113 A1
Figure 12B cont'd. Patent Application Publication May 19, 2016 Sheet 61 of 77 US 2016/0138113 A1
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FIGURE 12E Patent Application Publication May 19, 2016 Sheet 63 of 77 US 2016/0138113 A1
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FIGURE 13B Patent Application Publication May 19, 2016 Sheet 64 of 77 US 2016/0138113 A1
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FIGURE 13C Patent Application Publication May 19, 2016 Sheet 65 of 77 US 2016/0138113 A1
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FIGURE 13M Patent Application Publication May 19, 2016 Sheet 72 of 77 US 2016/0138113 A1
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FIGURE 14B Patent Application Publication May 19, 2016 Sheet 74 of 77 US 2016/0138113 A1
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FIGURE 14D Patent Application Publication May 19, 2016 Sheet 76 of 77 US 2016/0138113 A1
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METHODS AND COMPOSITIONS FOR THE most cancers and cancer treatments, there exist Subsets of DIAGNOSIS, PROGNOSIS AND TREATMENT patients that will respond to a particular treatment and Subsets OF BRAIN METASTASS of patients that will fail to respond to the treatment. 0008. The use of therapeutic predictive markers to identify 0001. This application claims the benefit of U.S. provi subsets of patients likely to respond to treatment would facili sional application Ser. No. 61/836,993 filed Jun. 19, 2013, the tate the selection of the appropriate treatment and avoid contents of which are hereby incorporated by reference into unnecessary delays associated with ineffective treatment. the instant application. Additionally, because most cancer treatments are associated FEDERALLY SPONSORED RESEARCHOR with adverse side effects inherent to the treatment, said pre DEVELOPMENT dictive markers eliminate unnecessary risks of adverse side effects by reducing the administration of cancer treatments to 0002 This invention was made with Government support individuals for whom treatment is likely to fail. under contract CA148967 and CA126518 awarded by the 0009 Metastasis is a complex series of steps in which National Cancer Institute/National Institutes of Health. The neoplasic cells leave the original tumor site and migrate to U.S. Government has certain rights in the invention. other parts of the body via the blood stream or the lymphatic system and start new tumors that resemble the primary tumor. FIELD OF THE INVENTION Breast cancer cells are often transported through the lym phatic pathway to bone or other areas Such as liver, lung or 0003. The invention described herein relates to methods brain. It may be life saving to predict whether a primary useful in the diagnosis, treatment and management of can cancer has the potential to metastasize such that high risk cers. In particular, the present invention relates to predicting patients can be subject to closer follow up or specific treat the likelihood of metastasis of a cancer to the brain, bone ment regime that will vary where the cancer has metastasized. and/or lung and its impact on metastasis-free Survival. Currently there is a need in the art for new and improved means by which to identify when a primary tumor, for BACKGROUND OF THE INVENTION example a breast cancer, is going to metastasize and how one 0004. After cardiovascular disease, cancer is the leading can inhibit the metastasis from the primary tumor to, for cause of death in the developed world. In the United States example, the brain, bone or lung of the patient. alone, over one million people are diagnosed with cancer 0010 Breast cancer is the most common cancer, and the each year, and over 500,000 people die each year as a result of Second leading cause of cancer death, among Women in the it. It is estimated that 1 in 3 Americans will develop cancer western world. It is the most common cancer in women and during their lifetime, and one in five will die from cancer. makes up a third of cancer occurrence of women in the US. Further, it is predicted that cancer may surpass cardiovascular Common tests that provide information to assists in the diag diseases as the number one cause of death within 5 years. As nosis or prognosis of breast cancer include mammograms and Such, considerable efforts are directed at improving treatment tissue biopsy followed by combinations of histological and diagnosis of this disease. examination, immune-histochemical detection with antibod 0005 Most cancer patients are not killed by their primary ies to estrogen receptor (ER), progesterone receptor (PR) tumor. They succumb instead to metastases: multiple wide and/or HER2/neu proteins. spread tumor colonies established by malignant cells that 0011 Currently, the recommended therapeutic predictive detach themselves from the original tumor and travel through markers in oncology are ER (estrogen receptor) and PR the body, often to distant sites. (progesterone receptor) status for selecting hormone sensi 0006 Cancer cells in an aggressive primary tumor are tive breast cancers, and HERB-2 for identifying breast cancer adept in exploiting that particular local tissue microenviron patients who may benefit from trastuzumab treatment. ment. In contrast, when metastatic cells leave these favorable 0012. The incidence of brain metastasis in patients with Surroundings, they must possess or acquire traits that will breast cancer overexpressing HERB-2 treated with trastu allow them to survive and colonize foreign, potentially hostile Zumab is twice that in other breast cancer patients. On the tissue environments. The obstacles that metastasizing tumor other hand, one-third of the patients with breast cancer will cells encounter vary from organ to organ, and are influenced develop CNS metastasis and this often occurs when they are by non-cancerous stromal cells of the tumor microenviron responding to therapy at other sites or have a stable disease. ment. For example, the blood-brain barrier, composed of Thus, drugs with a high impact on the clinical outcome of endothelial cells, astrocytes and pericytes, presents a far more metastatic breast cancer patients, such as taxanes or trastu formidable structure for tumor cells to penetrate, compared to Zumab, play only a limited role in the treatment of brain the fenestrated capillaries in the bone marrow. Tumor cells metastasis. with the capacity to extravasate and seed these different tissue 0013 Cerebral metastases occur in 10-15% of breast can microenvironments then encounter distinct cell types, often cer patients with advanced disease and have recently become with specialized functions, that can positively or negatively a significant clinical problem. It can be assumed that up to regulate Subsequent metastatic outgrowth. Indeed, dissemi 30% of metastatic breast cancer patients will experience brain nation can occur to multiple organs, yet metastatic tumors metastasis during the course of their disease. The increase in may grow in only one or a few sites, indicating critical roles this rate could be linked to greater survival in patients receiv for the microenvironment in this process. ing chemotherapy and the fact that it is difficult to overcome 0007 Clinical management of cancer can be aided by the blood brain barrier (BBB) with current systemic treat prognosis markers and by therapeutic predictive markers. ments. The difficulties in managing brain metastasis therapy Prognosis markers assess risk of the disease progression inde result in a median survival of seven months, with brain pendent of therapy. Therapeutic predictive markers indicate metastasis being the cause of death or a major contributing sensibility or resistance of a cancerto a specific treatment. For factor of it in 68% of patients. US 2016/0138113 A1 May 19, 2016
0014. An adequate estimation of independent predictive 0020 (ii) predicting that the subject will develop factors at initial tumor diagnosis is required to enable the metastasis to brain and bone if expression of PSMD11 is clinician to determine whether said tumor can metastasize. increased over control; This information would be useful for the clinician in order to 0021 (iii) predicting that the subject will develop decide between aggressive treatments, to avoid unnecessary metastasis to brain and lung if expression of one or more treatment, and to design therapies specifically addressed of SERPINB3, PI3, ADAMDEC1, ILF2, PSMB4, APP, against differential aspects of each metastatic location. S100A10, CTSC, CTSL1, CANX, ANXA5, PSMD2 Therefore, there is the need of predictive markers which and CTSB is increased over control, but will not develop provides information about the risk of metastasizing a pri metastasis to the brain if TPSG1 is increased over con mary tumor to other organs in order to treat efficiently the trol; illness. 0022 (iv) predicting that the subject will develop 0015. A number of strategies have been used to investigate metastasis to bone and lung if expression of one or more the constituent cell types of different tumor microenviron of MME, PSMB3, and PSMD10 is increased over con ments, predominantly in primary tumors, including cell sort trol; ing or laser capture microdissection followed by mRNA or 0023 (v) predicting that the subject will develop miRNA expression profiling. These approaches have led to metastasis to brain only if expression of one or more of the identification of expression signatures for tumor-associ SPOCK2, PSMB6, PRSS22, CTSS, KLK10, GZMK, ated macrophages, endothelial cells, fibroblasts, Tie2-ex ELANE, COX411, and TIMP2 is increased overcontrol, pressing monocytes, and astrocytes among others. While but will not developmetastasis to the brain if HNRPNPC these studies have been informative in identifying stromal and/or SEPT2 is increased over control; gene signatures, often with prognostic value, they involved 0024 (vi) predicting that the subject will develop manipulation of the tumor to isolate individual cell types, and metastasis to bone if expression of SNRNP200 is in most cases the stromal cells were isolated in isolation, increased over control, but will not develop metastasis to without comparative expression information for the tumor the bone if one or more of EIF3F, RPS6, GZMB, RPS13, cells. Thus, this information is not as informative as one RPS10, RPL21, RPL30, OAZ1, SerpinF2, RPL27, would desire. Accordingly, there is also a need in the art to PRTN3, RPS5, F2, RPL14, PSMD13, RPL28, RPS27A, understand the interplay between cancer cells and the TIMP1, RPS11, USP4, 1RPS24, CELA2B, and RPL11 microenvironment in intact tumors at different stages of is increased over control; metastatic seeding and outgrowth, and for better composi 0.025 (vii) predicting that the subject will develop tions and methods that relate to the manipulation of the meta metastasis to lung only if expression of one or more of static seeding and outgrowth process. PSMA7, TMPRSS5, F12, PSMA6, SPINK2, PSMA3, ADAM9, PLAU, AAPN3, ZNF146, ANXA1, PSMC2, SUMMARY OF THE INVENTION COPS7B, PSMB5, PSMC3, ANXA3, PSMA4, USP1, 0016. The inventors of the instant application set out to KIFAP3, PSMD4, HSP90AB1, PCSK1N, PSMB7, specifically analyze the interplay between cancer cells and PSMC1, ILF2, PSMD1, GDI2, and SERPINE2 is the microenvironment in intact tumors at different stages of increased over control, but will not develop metastasis to metastatic seeding and outgrowth. The inventors investigated the lung if one or more of SPINK4, ANXA9, PLAT, breast cancer cell interactions with the stroma in three organ MMP24, CST3, EEF2, F7, F10, RPL9, PRSS23, sites to which these cells commonly metastasize: the lung, MMP26, and HTRA1 is increased over control. bone and brain. 0026. In one embodiment, the cancer is breast cancer and 0017. One aspect of the present disclosure is directed to a the sample to be interrogated for gene expression is a cell or method of predicting the likelihood that a patient with cancer tissue sample from the primary tumor or bodily fluid which will develop metastasis to the brain, bone and/or lung, said may contain tumor cells. method comprising: (a) detecting in a sample from the Subject 0027. Another aspect of the present disclosure is directed the level of expression of genes SERPINB3, PI3, SPOCK2, to a method of predicting metastasis of breast cancer to the PSMB6, PRSS22, CTSS, KLK10, GZMK, ADAMDEC1, brain, bone and/or lung of a patient Suffering from breast ELANE, ILF2, PSMD11, PSMB4, S100A10, APP, COX4I1, cancer, said method comprising: isolating a sample from the CTSC, CTSL1, TIMP2, HNRPNPC, SEPT2, EIF3F, RPS5, patient; analyzing the sample for the increased expression of GZMB, RPS13, RPS10, RPL21, RPL30, OAZ1, SerpinF2, cathepsin S gene; and (i) predicting the breast cancer patient RPL27, PRTN3, RPS6, F2, RPL14, PSMD13, RPL28, has or is at risk of developing metastasis to the brain if there RPS27A, TIMP1, RPS11, USP4, RPS24, CELA2B, RPL11, is increased expression of cathepsin S gene in tumor cells SPINK4, ANXA9, PLAT, MMP24, CST3, EEF2, F7, F10, early on in brain metastasis development, relative to control; RPL9, PRSS23, MMP26, HTRA1, CANX, SLPI, ANXA5, and/or (ii) wherein, increased expression of cathepsin S gene PSMD2, CTSB, MME, PSMB3, SNRNP200, SLPI, in tumor cells early on in brain metastasis development, rela PSMD10, PSMA7, TMPRSS5, F12, PSMA6, SPINK2, tive to control, does not correlate with metastasis of the PSMA3, ADAM9, PLAU, AAPN3, ZNF146, ANXA1, patient's breast cancer to the patient's bone or lung. PSMC2, COPS7B, PSMB5, CTSB, PSMC3, ANXA3, 0028. One aspect of the present disclosure is directed to a PSMA4, USP1, KIFAP3, PSMD4, HSP90AB1, PCSK1N, method of treating, preventing or managing metastasis of CSTB, PSMB7, PSMC1, PSMD1, GDI2, SERPINE2, cancer cells from a primary tumor in a cancer patient to the TPSG1, PSMD2 and PSME1; and patient's brain, said method comprising: administering to 0018 (b) said patient an agent which inhibits cathepsin S. In one 0019 (i) predicting that the subject will develop embodiment, the primary cancer is breast cancer. In another metastasis to the brain, bone and lung if expression of embodiment, the agent is a selective inhibitor of cathepsin S. SLPI is increased over control; In a particular embodiment, the agent is a specific inhibitor of US 2016/0138113 A1 May 19, 2016 cathepsin S. The agent is, in one example, a peptide-based prising the steps of: (a) extracting the cell; (b) measuring an inhibitor of cathepsin S, which is based upon a peptide amount of cathepsin S, PSMB6, PSMD11 or SLPI nucleic sequence which comprises 2-20 consecutive residues of a acid expression or polypeptide in the cell; and (c) comparing preferred invariant chain cleavage site of cathepsin S. In one the amount of cathepsin S, PSMB6, PSMD11 or SLPI nucleic embodiment, the agent is administered to the patient Suffering acid expression or protein present in the cell to the amount of from cancer via intravenous injection, intradermal injection, cathepsin S, PSMB6, PSMD11 or SLPI nucleic acid expres Subcutaneous injection, intramuscular injection, intraperito sion or polypeptide in a sample isolated from normal, non neal injection, anal Supposition, vaginal Supposition, oral cancerous cells, wherein: (i) an amplified amount of cathep ingestion or inhalation. sin S and PSMB6 nucleic acid expression or polypeptide in 0029. One or more cathepsin S inhibitors are, in one the cell relative to the amount of cathepsin S and PSMB6 example, administered early on in the metastasis develop nucleic acid expression or polypeptide in the sample isolated ment cascade. In one embodiment, the peptide-based inhibi from normal, non-cancerous cells indicates that cancer is tor of cathepsin S is morpholinurea-leucine-homophenylala likely to metastasize to the brain, and/or (ii) an amplified nine-vinylsulfone phenyl (LHVS). In one embodiment, the amount of PSMD11 and SLPI nucleic acid expression or peptide-based inhibitor is a peptide-based vinylsulfone or a polypeptide in the cell relative to the amount of PSMD11 and modified peptide-based vinylsulfone. In another embodi SLPI nucleic acid expression or polypeptide in the sample ment, the peptide-based inhibitor is selected from peptidyl isolated from normal, non-cancerous cells indicates that can aldehydes, nitriles, C.-ketocarbonyls, halomethyl ketones, cer is likely to metastasize to the bone. diazomethyl ketones, (acyloxy)-methyl ketones, vinyl Sul 0034. In one embodiment, the cell is isolated from the fones, ketomethylsulfonium salts, epoxides, and N-peptidyl patient’s blood, or primary tumor. In another embodiment, O-acyl-hydroxylamines. In another embodiment, the agent is the cell is isolated from a primary breast tumor. selected from Asn-Leu-vinylsulfone, Arg-Met-vinylsulfone, 0035. In one aspect, the present invention is directed to a Leu-Arg-Met-Vinylsulfone, Glu-Asn-Leu-Vinylsulfone, and kit for determining in a sample from a subject with cancer Leu-Leu-Leu-Vinylsulfone. In one embodiment, the agent is expression levels of genes indicative of metastasis of cancer selected from N-(carboxybenzyl)-Asn-Leu-vinylsulfone, in the Subject to brain, bone or lung, the kit comprising one or N-(carboxybenzyl)-Arg-Met-vinylsulfone, N-(carboxyben more components for determining the expression levels of Zyl)-Leu-Arg-Met-vinylsulfone, N-(carboxybenzyl)-Glu said genes, wherein said one or more components are selected Asn-Leu-Vinylsulfone, and N-(carboxybenzyl)-Leu-Leu from the group consisting of a DNA array chip, an oligo Leu-vinylsulfone. nucleotide array chip, a protein array chip, an antibody, a 0030. Another aspect of the present disclosure is directed plurality of probes; and a set of primers for genes, SER to a method of treating, preventing or managing cancer cell PINB3, PI3, SPOCK2, PSMB6, PRSS22, CTSS, KLK10, metastasis in a cancer patient, comprising: extracting a GZMK, ADAMDEC1, ELANE, ILF2, PSMD11, PSMB4, sample from the primary tumor, metastatic tumor, or blood of S100A10, APP, COX411, CTSC, CTSL1, TIMP2, HNRP the cancer patient; assaying the sample to determine the NPC, SEPT2, EIF3F, RPS5, GZMB, RPS13, RPS10, RPL21, expression of cathepsin S and/or PSMB6 genes in said RPL30, OAZ1, SerpinF2, RPL27, PRTN3, RPS6, F2, sample; and administering a cathepsin S inhibitor if the RPL14, PSMD13, RPL28, RPS27A, TIMP1, RPS11, USP4, expression of cathepsin S and/or PSMB6 genes is increased RPS24, CELA2B, RPL11, SPINK4, ANXA9, PLAT, over control. MMP24, CST3, EEF2, F7, F10, RPL9, PRSS23, MMP26, 0031 One aspect of the present disclosure is directed to a HTRA1, CANX, SLPI, ANXA5, PSMD2, CTSB, MME, method for preparing a personalized genomics profile for a PSMB3, SNRNP200, SLPI, PSMD10, PSMA7, TMPRSS5, patient with breast cancer, comprising: extracting mono F12, PSMA6, SPINK2, PSMA3, ADAM9, PLAU, AAPN3, nuclear cells or cancer cells from the primary tumor and ZNF146, ANXA1, PSMC2, COPS7B, PSMB5, CTSB, Subjecting them to gene expression analysis; assaying the PSMC3, ANXA3, PSMA4, USP1, KIFAP3, PSMD4, sample to determine the expression of cathepsin S and HSP90AB1, PCSK1N, PSMB7, PSMC1, PSMD1, GDI2, PSMB6 in said sample; and generating a report of the data SERPINE2, PSMD2 and PSME1; each as set forth in Tables obtained by the expression analysis, wherein the report com 1 and 2. prises a prediction of the likelihood of the patient being sub 0036. In one aspect, the invention relates to use of a kit of stantially free of metastasis to the brain if, in addition to the invention for determining the risk of metastasis of cancer decreased expression of cathepsin S in the sample over con to the brain in a cancer patient. trol, expression of PSMB6 gene is also decreased over con 0037. In another aspect, a kit of the invention further com trol. In one embodiment, the method further comprises pre prises one or more reagents for RNA extraction; one or more dicting that the patient with cancer will develop metastasis to enzymes for syntheses of cDNA and cRNA; one or more the bone if in the sample over control, expression of PSMD11 reagents for hybridization for DNA chip, oligonucleotide or SLPI gene is increased over control. chip, protein chip, western blot, probes, or primers; one or 0032. In one aspect, the present disclosure is a kit for more reagents for binding of said antibodies to proteins determining treatment of a patient with brain metastasis, the indicative of recurrence of cancer; or DNA fragments of kit comprising means for detecting expression and/or activity control genes. of cathepsin Sand/or PSMB6 genes at an early stage of brain 0038. In another aspect, a kit of the invention further metastasis; and instructions for recommended treatment includes instructions for determining the likelihood of based on the presence of increased expression or activity in metastasis-free Survival for a patient based on the expression cathepsin S and/or PSMB6 genes. levels of the genes indicative of cancer metastasis. 0033. One aspect of the present disclosure is a method of 0039. In another aspect, the invention relates to a set of analyzing a cell expression profile for determining whether primers consisting of at least one primer pair for each of genes the cell is metastatic to the brain or bone, said method com SERPINB3, PI3, SPOCK2, PSMB6, PRSS22, CTSS, US 2016/0138113 A1 May 19, 2016
KLK10, GZMK, ADAMDEC1, ELANE, ILF2, PSMD11, experimental brain metastases, and cathepsin S expression in PSMB4, S100A10, APP, COX411, CTSC, CTSL1, TIMP2, primary breast tumors is inversely correlated with brain HNRPNPC, SEPT2, EIF3F, RPS5, GZMB, RPS13, RPS10, metastasis-free survival in patients. (a) Cross-species scatter RPL21, RPL30, OAZ1, SerpinF2, RPL27, PRTN3, RPS6, plot shows log-fold expression changes in the tumor and F2, RPL14, PSMD13, RPL28, RPS27A, TIMP1, RPS11, stromal gene space during the transition from early- to late USP4, RPS24, CELA2B, RPL11, SPINK4, ANXA9, PLAT, stage metastases in the brain. Genes that are differentially MMP24, CST3, EEF2, F7, F10, RPL9, PRSS23, MMP26, expressed only in the stroma (mouse) or in the tumor (human) HTRA1, CANX, SLPI, ANXA5, PSMD2, CTSB, MME, gene space are shown in pink or black respectively. Genes that PSMB3, SNRNP200, SLPI, PSMD10, PSMA7, TMPRSS5, are differentially expressed in both the stromal and tumor F12, PSMA6, SPINK2, PSMA3, ADAM9, PLAU, AAPN3, gene space are shown in purple. Grey dots represent ZNF146, ANXA1, PSMC2, COPS7B, PSMB5, PSMC3, homologs with either insufficient fold change or P values. ANXA3, PSMA4, USP1, KIFAP3, PSMD4, HSP90AB1, Horizontal and vertical lines denote fold change cut-off for PCSK1N, CSTB, PSMB7, PSMC1, PSMD1, GDI2, SER significance. Genes in the lower right quadrant (CTSS and PINE2, PSMD2 and PSME1. CST7) represent genes that are downregulated in tumor cells 0040. In another aspect, the invention relates to an array in late metastases, and concomitantly upregulated in stromal consisting of a substrate or solid support and at least one cells. (b) Expression of tumor-derived (human) and stromal probe for each of genes SERPINB3, PI3, SPOCK2, PSMB6, derived (mouse) cathepsin S (CTSS or Ctss respectively) in PRSS22, CTSS, KLK10, GZMK, ADAMDEC1, ELANE, Br-M control (Ctrl; n=11) cell line, normal brain (n=12), and ILF2, PSMD11, PSMB4, S100A10, APP, COX411, CTSC, early-/late-stage brain metastases (classified by BLI inten CTSL1, TIMP2, HNRPNPC, SEPT2, EIF3F, RPS5, GZMB, sity; n=16 for early-stage and n=17 for late-stage metastases) RPS13, RPS10, RPL21, RPL30, OAZ1, SerpinF2, RPL27, from Athy/nu mice. mRNA expression is depicted relative to PRTN3, RPS6, F2, RPL14, PSMD13, RPL28, RPS27A, the Br-MCtrl cell line for human CTSS and relative to normal TIMP1, RPS11, USP4, RPS24, CELA2B, RPL11, SPINK4, brain for mouse Ctss. All assays were performed in triplicate ANXA9, PLAT, MMP24, CST3. EEF2, F7, F10, RPL9, and gene expression was normalized to Ubc for all stromal PRSS23, MMP26, HTRA1, CANX, SLPI, ANXA5, PSMD2, genes (mouse origin). B2M for tumor cell-derived genes (hu CTSB, MME, PSMB3, SNRNP200, SLPI, PSMD10, man origin). (c) Metastasis-free survival (MFS) for breast PSMA7, TMPRSS5, F12, PSMA6, SPINK2, PSMA3, cancer patients (GSE 12276 data set) based on low, medium ADAM9, PLAU, AAPN3, ZNF146, ANXA1, PSMC2, and high CTSS expression at the primary site. The Kaplan COPS7B, PSMB5, PSMC3, ANXA3, PSMA4, USP1, Meier plot demonstrates that high CTSS expression is KIFAP3, PSMD4, HSP90AB1, PCSK1N, CSTB, PSMB7, inversely correlated with brain MFS, while there is no such PSMC1, PSMD1, GDI2, SERPINE2, PSMD2 and PSME1. association for bone or lung. (d) Representative images of matched primary breast cancer and brain metastasis patient BRIEF DESCRIPTION OF THE DRAWINGS samples stained for CTSS (red) and the macrophage marker 0041 FIG. 1 shows that HuMuProtIn array enables simul CD68 (white), or pan-cytokeratin (CK; green) to visualize taneous acquisition of gene expression changes in tumor and tumor cells. DAPI staining was used to visualize cell nuclei stromal cells. (A) is a schematic of the experimental design (blue). Scale bar indicates 50 pum. (e) Quantification of pro employed to analyze tumor stroma interactions in different portions of tumor cells and macrophages, presented as the metastatic microenvironments. Br-M-brain metastatic, percentage of total DAPI cells, in matched primary breast Bo-M-bone metastatic, and Lu-M lung metastatic variants cancer and brain metastases samples. (f) Quantification of the of the human breast cancer line MDA-MB-231 cell line were CTSS index as a measure of relative CTSS levels in tumor injected intracardially or intravenously into immunocompro cells and macrophages. Data are presented as bars--S.e.m. or mised mice. RNA was isolated from whole brain-, bone-, and as box plots with whiskers to illustrate minimum and maxi lung metastases that contain human tumor cells and mouse mum values. The horizontal line depicts the mean. P values stromal cells. Gene expression changes in Xenografted ani were obtained using two-tailed unpaired t-test for (b) and a mals were analyzed with the dual-species specific array log-rank test for (c). *P<0.01, and ***P<0.001. HuMu ProtIn (Human/Murine Proteases and Inhibitors). (B) 0.043 FIG.3 illustrates that macrophages are the predomi Principal componentanalysis of the HuMu array data: the 1st nant source of stromal-derived cathepsin S and only com and 2nd components are plotted on the X and y axes respec bined depletion of tumor- and stromal-derived cathepsin S tively. These components together represent the largest reduces experimental brain metastasis. (a) Representative sources of variation in the dataset. The first and second com images of normal brain, early- and late-stage brain metastasis ponents represent 89.98% and 8.44% respectively of the vari (classified by BLI intensity) co-stained for Ctss/CTSS (red) ance in the tumor gene space, and 90.83% and 4.06% in the and GFP (tumor cells; green) or Ibal (macrophages/micro stromal gene space. This analysis revealed variation in tumor glia; white). Tumor cell-derived CTSS is indicated by the gene expression that was associated with differences between arrowhead and macrophage-derived Ctss is indicated by the early- and late-stage metastasis. Meanwhile, variation in the arrow. (b) Kaplan-Meier curve shows the percentage of brain stroma was associated with both stage and tissue. Dotted metastasis-free animals in the 4 experimental groups indi ellipses were drawn manually to indicate related data points cated in the table. Ctrl; Ctss WT(n=21 mice), CTSS KD; Ctss within stage or organ. (C, D) Heatmaps of (C) tumor- and (D) WT (n=16), Ctrl; Ctss KO (n=22), and CTSS KD; Ctss KO stroma-derived genes that were differentially expressed (n=12). (c) Quantification of the ex vivo BLI intensity on day between early and late metastases across different organ sites. 35 after Br-M tumor cell inoculation. Ctrl, Ctss WT (n=10), The lung stroma did not show extensive differences between CTSS KD; Ctss WT (n=7), Ctrl; Ctss KO (n=13), and CTSS early and late stages (Table 1 g). KD; Ctss KO (n=11). (d) Representative ex vivo BLI images 0042 FIG. 2 shows that cathepsin S shows highly regu of the 4 experimental groups as shown in (c). (e) Quantifica lated stage- and cell type-specific expression changes in tion of tumor cell proliferation (percentage of Ki67"GFP" US 2016/0138113 A1 May 19, 2016
cells) on day 35 after tumor cell inoculation. Ctrl, Ctss WT HBMECs. (c) Representative images of control brain, bone, (n=8), CTSS KD; Ctss WT (n=8), Ctrl; Ctss KO (n=6), and and lung sections stained for the tight junction proteins Jam CTSS KD; Ctss KO (n=10). Scale bar indicates 50 B, Ocln or Cldn3 (white), with CD31 (red) to visualize blood Circles represent individual mice and horizontal lines repre vessels. DAPI was used as a nuclear counterstain. (d) Sche sent the meants.e.m. P values were obtained with Mantel matic of the cell-based cleavage assay. (e) Western blot analy Cox log-rank test for MFS and with two-tailed unpaired t-test sis showing increased JAM-B in HBMEC-conditioned media for numerical data. *P<0.05, **P<0.01 and ***P<0.001. (CM) after incubation with Br-M cell CM for the indicated 0044 FIG. 4 shows that cathepsin S deficiency in tumor time points. Addition of the cathepsin S specific inhibitor cells and macrophages impairs metastatic seeding and out VBY-999 (10 uM) resulted in reduced accumulation of growth. (a) Representative images of brain metastases (day JAM-B in HBMECCM at the indicated time points. Incuba 35) stained for GFP (tumor cells; green), the endothelial cell tion with PBS pH 6.0, 0.05 mM DTT served as a control for marker CD34 (white), and DAPI to visualize nuclei (blue). baseline JAM-B shedding of HBMEC. Each western blot Scale bar indicates 50 Lum. (b) GFP' tumor cells were catego shows the representative result of three independent experi rized based on their localization relative to blood vessels, ments. Scale bar indicates 20 Jum. Graphs represent mean+S. defined as the distance of tumor cells from blood vessels (1 to e.m. P values were obtained using two-tailed unpaired t-test. >4 average tumor cell diameter), and the percentage of tumor NS=not significant, ***P<0.001. cells in each defined area was quantified using Metamorph 0047 FIG. 7 shows that pharmacological inhibition of image analysis software. Ctrl, Ctss WT (n=4), CTSS KD; cathepsin S reduces brain metastasis formation in a preclini Ctss WT (n=6), Ctrl; Ctss KO (n=6), and CTSS KD; Ctss KO cal trial. (A) Schematic of the prevention trial experimental (n-6). Categorical data are plotted as stacked bars. P values design. (B) Quantification of VBY-999 concentrations in were obtained with an ordinal Chi-square test for categorical plasma and brain tissue at the indicated time points after data. **PCO.O1 and ***PCO.OO1. treatment started (n=3 for each group). (C) Quantification of 004.5 FIG. 5 Cathepsin S mediates blood-brain barrier BLI intensity in the head region at the indicated time points transmigration of brain metastatic cells. (a) Quantification of after Br-M cell inoculation. n=20 for vehicle group (5% dex BLI intensity at the indicated time points relative to BLI trose in water (D5W)) and n=21 for VBY-999 treatment signal immediately after tumor cell inoculation. Ctrl, CtSS group (100 mg/kg/day). The BLI signal in the VBY-999 ver WT (n=10), CTSS KD; Ctss WT (n=9), Ctrl, Ctss KO (n=8), sus control group is 77, 70 and 65% reduction at each of the and CTSS KD; Ctss KO (n=8) for the 24 h time point, and n=5 three successive time points indicated. (D) Representative for each group for the 48 h time point. (b) Representative BLI BLI images at the trial endpoint, d35 after Br-M cell inocu images in the 4 experimental groups immediately (Oh) and 48 lation. (E) Quantification of BLI intensity at d35 after Bo-M h after tumor cell injection in vivo (top panels) and ex vivo tumor cell inoculation in the bone and spine region. Vehicle (lower panel). (c) Tumor cells were categorized based on their (n=12 mice) and VBY-999 (n=13). (F) Representative BLI localization relative to the vasculature defined as intravascu and X-ray images at day 35 after Bo-M cell inoculation. lar, extravasating and extravascular, and the percentage of Arrows indicate osteolytic lesions. Bars represent mean+S.e. viable tumor cells in each category was quantified at the m. for (b), circles represent individual mice and horizontal indicated time points. Ctrl; Ctss WT (n=4), CTSS KD; Ctss lines represent the meants.e.m for (C, E). P values were WT (n=3), Ctrl; Ctss KO (n=3), and CTSS KD; Ctss KO obtained using two-tailed unpaired t-test. NS not significant, (n=4) for the 24 h time point, and n=4 for each group for the * P&O.05. 48 h time point. (d) Quantification of the number of transmi 0048 FIG. 8 illustrates characterization of the stromal cell grated Br-M Ctrl and CTSS KD cells in the presence or types in early- and late-stage brain, bone and lung metastasis. absence of the cathepsin S-specific inhibitor VBY-999 (A-C) Quantification and representative images of the in vivo through an in vitro BBB formed with human brain microvas BLI intensity are shown for (a) brain metastases, (B) bone cular endothelial cells (HBMEC) in co-culture with astro metastases, and (C) lung metastases for early and late stages. cytes. Circles represent individual mice and horizontal lines Circles represent individual mice and horizontal lines repre represent the meants.e.m. for numerical data shown in (a). sent the meants.e.m. (D-F) Representative images of control Graphs represent mean+S.e.m in (d). Categorical data are tissue and early- and late-stage brain, bone, and lung plotted as stacked bars. P values were obtained with two metastases stained for GFP (tumor cells) and the endothelial tailed unpaired t-test for numerical data and with an ordinal marker CD34 or the macrophage markers CD68 (lung and Chi-square test for categorical data. NS not significant, bone) or Iba1 (brain). DAPI staining was used to visualize cell *P<0.05, **P<0.01, and ***P<0.001. nuclei. Brain metastasis sections were also stained with the 0046 FIG. 6 shows that cathepsin S cleaves tight junction astrocyte-specific marker GFAP. Scale bar indicates 50 Lum. proteins that regulate blood-brain barrier integrity. (a) West 0049 FIG.9 demonstrates tissue- and stage-specific gene ern blot analysis of CTSS-mediated cleavage of recombinant expression changes in tumor and stroma. (A) Venn diagram of tight junction proteins (junctional adhesion molecules the tumor-derived genes that are significantly different (JAM)-A, -B and -C, occludin (OCLN), claudins (CLDN)-3 between early and late metastases across different metastatic and -5), and adherens junction proteins cadherin 5 (CDH5) sites (FIG. 1c). Of the 308 genes significantly different and CD31 for the indicated time points at pH 4.5 and pH 6.0 between early and late stage in either brain, bone or lung in the presence or absence of the cathepsin S-specific inhibi metastases, 176 genes change by stage in all three sites. (B) tor VBY-999. VBY-999 was used at 10 uM, a concentration Venn diagram depicting the overlap of the 75 stroma-derived that efficiently inhibits cathepsin S. (b) mRNA expression of genes that are significantly different between early- and late the tight junction and adherens junction molecules in stage metastases across brain, bone and lung (FIG. 1D). HUVECs and HBMECs (n=9 for each cell line). All assays Unlike the tumor-derived genes depicted in (A), there were no were run in triplicate and gene expression was normalized to stromal-derived genes that were significantly different B2M. Expression is depicted relative to expression in between early- and late-stage metastases in all three tissues US 2016/0138113 A1 May 19, 2016 investigated. (C) Heatmap depicting tissue-specific gene resentative of three independent samples per stage. Scale bar expression in the brain, bone and lung Stroma. No tissue indicates 50 um. For qPCR validation: n=11, 15, 15, 13, 7, 15 specific genes were found for tumor-derived genes. Proteases samples in (f), n=11, 15, 15, 13, 7, 5 samples in (b), n=8, 15, are denoted in purple, endogenous inhibitors in red, and their 7, 9, 5, 15 samples in (g), n=8, 15, 7, 9, 5, 5 samples in (h), interacting partners in black. (D) qPCR confirmed the tissue n=8, 6, 15, 12, 5, 3 samples in (i). n=5 and 9 samples for enriched expression pattern of Htral for brain, Mmp 13 for control lung and late-stage metastases (Xenograft model), and bone, and Mmp11 for lung in control tissue, early- and late n=6 and 9 for control lung and late-stage metastases (synge stage metastases. Graphs represent mean+S.e.m. P values neic model). mRNA expression is depicted relative to early were obtained using two-tailed unpaired t-test. *P<0.05, stage metastases in(fi) and relative to control tissue in (). All **P<0.01, and ***P<0.001. assays were performed in triplicate and gene expression was 0050 FIG. 10 shows the independent validation of differ normalized to Ubc for all stromal genes, B2M for tumor entially expressed genes in experimental brain, bone and lung cell-derived genes. P values were obtained using two-tailed metastases. (a-e) Representative images of control (non unpaired t-test: NS=not significant, *P<0.05, *P<0.01, and tumor-burdened) tissue, early- and late-stage site-specific *** P&O.OO1. metastases (classified by BLI intensity as in FIG. 8) showing 0051 FIG. 11 shows the identification of genes associated immunofluorescence staining of tumor- and stromal-derived with metastasis-free survival (MFS) and differentially proteases and protease inhibitors exhibiting stage-dependent expressed between the tumor and stroma. (A-B) As shown in expression changes in the HuMu Protnarray. (a) Brain sec FIG.2a for brain metastasis, cross-species scatter plots depict tions were stained with antibodies against the protease CTSZ expression changes in the tumor and stromal gene space and the protease inhibitor TIMP2 as representative candidates during the transition from early- to late-stage metastasis for that were differentially expressed in tumor cells. (b) Bone (a) bone and (b) lung metastasis. Differentially expressed sections were stained with antibodies against the protease genes in the Stroma (mouse) or in the tumor (human) gene ADAM17 and the protease inhibitor SERPINB10 to repre space are shown in pink or black respectively. Genes that are sent differentially expressed candidates in tumor cells in bone differentially expressed in both the stroma and the tumor gene metastases. (c) Lung sections were stained with antibodies space are shown in purple. (C-E) Differentially expressed against the protease MMP24 and the protease inhibitor SER genes shown in FIG. 1c were analyzed for association with PINE2 to confirm stage-differential expression in tumor cells MFS for either (C) brain, (D) bone, or (E) lung metastasis, in lung metastases. (d) Staining for the stromal-derived pro depending on the tissue in which the gene was differentially tease Bace1 and the protease inhibitor Timp1 confirmed expressed. Scaled gene expression values were used in a Cox stage-specific expression changes in GFAP+ astrocytes. (e) proportional hazards model as described in the methods. Haz Staining for the protease Ctse and the protease inhibitor Csta ard ratios (HR) and 95% confidence intervals are shown for confirmed stage-specific stromal changes in bone metastasis. each organ site. HR <1.0 is associated with better patient CD68+ macrophages were identified as the predominant prognosis, whereas HR1.0 is associated with poor patient source for Cstain bone metastases. qPCR was used to confirm prognosis. (F) Genes depicted in (C-E) are shown in the Venn stage-differential expression changes, and to determine if the diagram, where few genes were found to be significantly expression changes in Stromal cells in brain or bone metasta associated with MFS in multiple tissues. A single gene, SLPI, sis are a general response to tumor cell colonization of the was significantly associated with MFS in all three tissues: respective organ, or if the expression changes depend on the high expression of levels of SLPI correlated with poor patient tumor cell variant. Hatched bars represent mismatched prognosis. Hazard ratio significance was determined using samples (BrM in bone and Bo-M in brain). Filled bars indi Wald’s test with a nominal P value cutoff of <0.05. cate the matched samples. (f-g) qPCR for Ctsh, Cst7, 0.052 FIG. 12 Tumor cells and macrophages are the major Pcskiln, Serpinil (brain) in (f) and Adamts4, Adamts 12, constituent cell types of patient brain metastases and express Casp2, Ctse (bone) in (g) confirmed stage-differential expres high levels of CTSS. (a-b) Representative images of (a) pri sion changes in a tissue-dependent manner. Mismatched mary breast cancer and (b) brain metastases patient samples samples did not show significant changes. (hi) qPCR for stained for CTSS (red) in combination with the macrophage ADAM21, CTSZ, FAU, TIMP2 (brain) in (h) and ADAM17, marker CD68 (white) or a pan-cytokeratin (CK) antibody to CASP3, DPP8 (bone) in (i) confirmed stage-differential visualize tumor cells (green). DAPI staining was used to expression changes in a tissue-dependent manner. Mis visualize cell nuclei (blue). The patient samples shown here matched samples (Br-M to bone and Bo-M to brain) revealed represent different subtypes of breast cancer based on ER/PR/ that tumor cells underwent the same significant expression Her2 status. Scale bar indicates 100 um in the upper panel changes between early and late stages as identified for rows and 20 um in the two lower panel rows. (c) Quantifica matched samples in the respective tissue. () qPCR for SER tion of proportions of CK+ tumor cells and CD68+ macroph PINE2 confirmed stage-differential expression changes in ages in brain metastases samples (these are from patients for Lu-M tumor cells in lung metastasis, while those changes which there was no matched primary breast tumor tissue were not present in Br-M and Bo-M cells in brain or bone available). (d) Combined quantification of proportions of metastasis, respectively. (k) qPCR Serpina3n and Timp1 con CK-- tumor cells, CD68+ macrophages, and the remaining firmed Stage-differential expression changes (control vs. late) CK-CD68- cell population in primary breast cancer (n-6) in the stroma of lungs from Xenografted animals as well as and brain metastasis samples (n=13; either matched to the lungs from the immunocompetent, syngeneic MMTV-PyMT primary, or unmatched samples). (e) Quantification of the breast cancer model. (1) Immunofluorescence staining for CTSS index as a measure of relative CTSS protein levels in Serpina3n and Timp1 in lungs of MMTV-PyMT breast cancer tumor cells and macrophages. Data in (c) and (e) are pre model (upper panels) and Ctss (red) in co-staining with GFP sented as box plots with whiskers to illustrate minimum and (tumor cells; green) or Ibal (macrophages; white) in the maximum values. The horizontal line depicts the mean. Data syngeneic PyMT-BrM model (lower panels). Images are rep in (d) are presented as stacked bars--S.e.m. US 2016/0138113 A1 May 19, 2016
0053 FIG. 13 shows that cathepsin S deficiency differen cific inhibitor VBY-999 through an invitro BBB formed with tially alters brain metastasis growth kinetics, but does not human umbilical vein endothelial cells (HUVECs) or human affect viability of Br-M cells or vessel formation in mice. (a) brain microvascular endothelial cells (HBMECs) in co-cul Quantification of CTSS mRNA expression in Br-MCtrl and ture with astrocytes and quantification of the number of trans Br-MCTSS KD tumor cell lines (n=7 replicates). Expression migrated Br-M cells through Transwell chambers on which is depicted relative to Br-MCtrl cells. All assays were run in HUVECs, HBMECs or astrocytes were either seeded as triplicate and gene expression was normalized to B2M. (b) monolayers, or controls in which none of these cells were Western blot analysis of CTSS expression levels in cell seeded. (c) Expression of tightjunction and adherens junction lysates and conditioned media (CM) from Br-M Ctrl and proteins was confirmed in an independent data set Br-M CTSS KD cells. Western blot shows representative (GSE47067) of FACS sorted endothelial cells. Only Jam-B result from 3 independent experiments. qPCR and western and OcIn are significantly enriched in brain endothelial cells blotting revealed a 90% knockdown efficiency for CTSS at compared to either lung or bone endothelial cells. (d) Western both the mRNA and protein level. (c) Representative images blot analysis of recombinant JAM-A, -B and -C cleavage by of CTSS immunofluorescence staining of Br-M Ctrl and CTSS, in the presence or absence of the specific inhibitor Br-MCTSS KD cell lines. DAPI staining was used to visu VBY-999 (10 uM), using an antibody that detects the IgG1 alize cell nuclei (blue). Scale bar indicates 20 lum. (d) CTSS domain in the recombinant JAM fusion proteins. (e) Align KD does not affect cell viability in culture as determined by ment of the amino acid sequence of JAM-A, -B, and -C. MTT assays (n=4 replicates). (e) Brain metastases size and Motifs that are conserved in all 3 family members are high vessel density were defined as the area covered by GFP (tu lighted in dark purple, and motifs that are conserved in 2 of mor cells) or CD34 (endothelial cells) respectively, and the the 3 JAM family members are depicted in light purple. The GFP-covered area was quantified relative to CD34-covered putative cleavage location for cathepsin S is indicated by the area using Metamorph image analysis. (f) Quantification of red box. (f) Quantification of the three independent JAM-B vessel density in the brain, defined as the ratio of Texas Red cell based cleavage experiments. Graphs represent meants. Lectin-- area to total DAPI area (n=4 for WT. n=6 for Ctss e.m. in (a) and (b), as box plots with whiskers to illustrate KO), and (g) assessment of vessel permeability by intrave minimum and maximum values in (c). The horizontal line neous injection of Evan's blue dye (n=8 for each group). (h) depicts the mean. Circles represent individual samples and Quantification of BLI intensity in vivo at the indicated time horizontal lines represent the meants.e.m. in (f). P values points after tumor cell inoculation. Ctrl, Ctss WT (n=16), were obtained using two-tailed unpaired t-test. NS=not sig CTSS KD; Ctss WT(n=15), Ctrl: Ctss KO (n=11), and CTSS nificant, *P<0.05, **P<0.01, and ***P<0.001. KD; Ctss KO (n=10). (i) Schematic of the regression trial experimental design. () Quantification of the BLI intensity DETAILED DESCRIPTION OF THE INVENTION from d0-d35 after Br-M tumor cell inoculation. At d27, mice 0055 All publications, patents and other references cited were stratified into vehicle and VBY-999 treatment groups herein are incorporated by reference in their entirety into the (n=7 per group) to achieve equal average BLI intensity at the present disclosure. time of treatment start at d28. Mice were dosed daily with 0056 To facilitate understanding of the invention, the fol either vehicle or VBY-999 (100 mg/kg) for 7 days and lowing definitions are provided. It is to be understood that, in metastasis growth was monitored by BLI imaging and general, terms are to be given their ordinary meaning or revealed no significant changes between vehicle and VBY meanings as generally accepted in the art unless otherwise 999 treated animals in an intervention trial. (k) Quantification indicated. The terminology used herein is for the purpose of of the BLI intensity in the bone and spine region of vehicle describing particular embodiments only and is not intended to treated and VBY-999-treated animals from the prevention limit the scope of the present invention which will be limited trial, at d35 following Br-M tumor cell inoculation (n=23 for only by the appended claims. vehicle and n=21 for VBY-999). (1) Quantification of the BLI 0057. In practicing the present invention, many conven intensity in the bone and spine region at d35 following Br-M tional techniques in molecular biology are used. These tech tumor cell inoculation, in the four experimental groups. Ctrl; niques are described in greater detailin, for example, Molecu Ctss WT (n=10), CTSS KD; Ctss WT (n=8), Ctrl; Ctss KO lar Cloning: a Laboratory Manual 3rd edition, J. F. Sambrook (n=10), and CTSS KD; Ctss KO (n=11). (m) Quantification of and D. W. Russell, ed. Cold Spring Harbor Laboratory Press the BLI intensity in the head region (which may arise from 2001 and DNA Microarrays: A Molecular Cloning Manual. skull and/or brain lesions) of vehicle-treated and VBY-999 D. Bowtell and J. Sambrook, eds. Cold Spring Harbor Labo treated animals from the prevention trial, at d35 following ratory Press 2002. Additionally, standard protocols, known to Bo-M tumor cell inoculation (n=12 for vehicle and n=13 for and used by those of skill in the art in mutational analysis of VBY-999). Graphs represent meants.e.m or circles represent mammalian cells, including manufacturers instruction individual mice and horizontal lines represent the meants.e. manuals for preparation of samples and use of microarray m. P values were obtained using two-tailed unpaired t-test. platforms are hereby incorporated by reference. NS=not significant, *P<0.05. 0058. In the description that follows, a number of terms 0054 FIG. 14 shows that cathepsin S deficiency impairs are used extensively. The following definitions are provided transmigration in an in vitro BBB assay, and sequence analy to facilitate understanding of the invention. Unless otherwise sis identifies a putative cleavage site for cathepsin S. (a) specified, “a,” “an.” “the and “at least one' are used inter Pharmacological inhibition of cathepsin S with increasing changeably and mean one or more than one. concentrations of the cathepsin S-specific inhibitor VBY-999 0059. The terms “cancer”, “cancerous”, or “malignant” (OuM (Vehicle) to 100 uM) did not affect Br-M cell viability refer to or describe the physiological condition in mammals as determined by MTT assays (n=3 replicates). (b) Quantifi that is typically characterized by unregulated growth of tumor cation of the number of transmigrated Br-M Ctrl and CTSS cells. Examples of a blood cancer include but are not limited KD cells in the presence or absence of the cathepsin S-spe to acute myeloid leukemia. US 2016/0138113 A1 May 19, 2016
0060. The term "diagnose' as used herein refers to the act sample RNA or DNA via complementary sequences (probes) or process of identifying or determining a disease or condi allows the determination of the level of gene expression in the tion in a mammal or the cause of a disease or condition by the sample tested. evaluation of the signs and symptoms of the disease or disor 0066. Therapeutic agents for practicing a method of the der. Usually, a diagnosis of a disease or disorder is based on present invention include, but are not limited to, inhibitors of the evaluation of one or more factors and/or symptoms that the expression or activity of genes identified and disclosed are indicative of the disease. That is, a diagnosis can be made herein, or protein translation thereof. An “inhibitor is any based on the presence, absence or amount of a factor which is Substance which retards or prevents a chemical or physiologi indicative of presence or absence of the disease or condition. cal reaction or response. Common inhibitors include but are Each factor or symptom that is considered to be indicative for not limited to antisense molecules, antibodies, and antago the diagnosis of a particular disease does not need be exclu nists. sively related to said particular disease; i.e. there may be differential diagnoses that can be inferred from a diagnostic 0067. The term “poor as used herein may be used inter factor or symptom. Likewise, there may be instances where a changeably with “unfavorable.” The term “good” as used factor or symptom that is indicative of a particular disease is herein may be referred to as “favorable.” The term “poor present in an individual that does not have the particular responder as used herein refers to an individual whose can disease. cer grows during or shortly thereafter standard therapy, for example radiation-chemotherapy, or who experiences a clini 0061 “Expression profile' as used herein may mean a cally evident decline attributable to the cancer. The term genomic expression profile. Profiles may be generated by any “respond to therapy' as used herein refers to an individual convenient means for determining a level of a nucleic acid whose tumor or cancer either remains stable or becomes sequence e.g. quantitative hybridization of microRNA, smaller/reduced during or shortly thereafter standard therapy, labeled microRNA, amplified microRNA, cRNA, etc., quan for example radiation-chemotherapy. titative PCR, ELISA for quantitation, and the like, and allow the analysis of differential gene expression between two 0068 “Probes’ may be derived from naturally occurring or recombinant single- or double-stranded nucleic acids or samples. A subject or patient tumor sample, e.g., cells or may be chemically synthesized. They are useful in detecting collections thereof, e.g., tissues, is assayed. Samples are col the presence of identical or similar sequences. Such probes lected by any method known in the art. may be labeled with reporter molecules using nick transla 0062. The term "expression product” or “gene expression tion, Klenow fill-in reaction, PCR or other methods well product as referred to herein may be a protein or a transcript known in the art. Nucleic acid probes may be used in south (i.e., an RNA molecule transcribed from the gene). ern, northern or in situ hybridizations to determine whether 0063 “Gene' as used herein may be a natural (e.g., DNA or RNA encoding a certain protein is present in a cell genomic) gene comprising transcriptional and/or transla type, tissue, or organ. tional regulatory sequences and/or a coding region and/or 0069. The term “prognosis' as used herein refers to a non-translated sequences (e.g., introns, 5'- and 3'-untrans prediction of the probable course and outcome of a clinical lated sequences). The coding region of a gene may be a condition or disease. A prognosis of a patient is usually made nucleotide sequence coding for an amino acid sequence or a by evaluating factors or symptoms of a disease that are indica functional RNA, such as tRNA, rRNA, catalytic RNA, skNA, tive of a favorable or unfavorable course or outcome of the miRNA or antisense RNA. The term “gene' has its meaning disease. The phrase “determining the prognosis' as used as understood in the art. However, it will be appreciated by herein refers to the process by which the skilled artisan can those of ordinary skill in the art that the term “gene' has a predict the course or outcome of a condition in a patient. The variety of meanings in the art, some of which include gene term “prognosis' does not refer to the ability to predict the regulatory sequences (e.g., promoters, enhancers, etc.) and/or course or outcome of a condition with 100% accuracy. intron sequences, and others of which are limited to coding Instead, the skilled artisan will understand that the term sequences. It will further be appreciated that definitions of “prognosis' refers to an increased probability that a certain 'gene include references to nucleic acids that do not encode course or outcome will occur; that is, that a course or outcome proteins but rather encode functional RNA molecules such as is more likely to occur in a patient exhibiting a given condi tRNAs. For the purpose of clarity we note that, as used in the tion, when compared to those individuals not exhibiting the present application, the term 'gene’ generally refers to a condition. A prognosis may be expressed as the amount of portion of a nucleic acid that encodes a protein; the term may time a patient can be expected to Survive. Alternatively, a optionally encompass regulatory sequences. This definition is prognosis may refer to the likelihood that the disease goes not intended to exclude application of the term “gene' to into remission or to the amount of time the disease can be non-protein coding expression units but rather to clarify that, expected to remain in remission. Prognosis can be expressed in most cases, the term as used in this document refers to a in various ways; for example prognosis can be expressed as a protein coding nucleic acid. percent chance that a patient will survive after one year, five years, ten years or the like. Alternatively prognosis may be 0064 “Mammal’ for purposes of treatment or therapy expressed as the number of months, on average, that a patient refers to any animal classified as a mammal, including can expect to Survive as a result of a condition or disease. The humans, domestic and farm animals, and Zoo, sports, or pet prognosis of a patient may be considered as an expression of animals, such as dogs, horses, cats, cows, etc. Preferably, the relativism, with many factors effecting the ultimate outcome. mammal is human. For example, for patients with certain conditions, prognosis 0065 “Microarray' refers to an ordered arrangement of can be appropriately expressed as the likelihood that a con hybridizable array elements, preferably polynucleotide dition may be treatable or curable, or the likelihood that a probes, on a substrate or solid support. Hybridization of disease will go into remission, whereas for patients with more US 2016/0138113 A1 May 19, 2016 severe conditions prognosis may be more appropriately 0077. A prognosis is often determined by examining one expressed as likelihood of survival for a specified period of or more prognostic factors or indicators. These are markers, time. the presence or amount of which in a patient (or a sample 0070. The term “relapse” or “recurrence” as used in the obtained from the patient) signal a probability that a given context of cancer in the present application refers to the return course or outcome will occur. The skilled artisan will under of signs and symptoms of cancer after a period of remission or stand that associating a prognostic indicator with a predispo improvement. sition to an adverse outcome may involve statistical analysis. 0071. As used herein a “response' to treatment may refer Additionally, a change in factor concentration from a baseline to any beneficial alteration in a Subject's condition that occurs level may be reflective of a patient prognosis, and the degree as a result of treatment. Such alteration may include stabili of change in marker level may be related to the severity of Zation of the condition (e.g., prevention of deterioration that adverse events. Statistical significance is often determined by would have taken place in the absence of the treatment), comparing two or more populations, and determining a con amelioration of symptoms of the condition, improvement in fidence interval and/or a p value. See, e.g., Dowdy and the prospects for cure of the condition. One may refer to a Wearden, Statistics for Research, John Wiley & Sons, New Subjects response or to a tumors response. In general these York, 1983. In one embodiment, confidence intervals of the concepts are used interchangeably herein. invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% 0072 “Treatment” or “therapy” refer to both therapeutic and 99.99%, while preferred p values are 0.1, 0.05, 0.025, treatment and prophylactic or preventative measures. The 0.02, 0.01, 0.005, 0.001, and 0.0001. Exemplary statistical term “therapeutically effective amount” refers to an amount tests for associating a prognostic indicator with a predisposi of a drug effective to treat a disease or disorder in a mammal. tion to an adverse outcome are described. In the case of cancer, the therapeutically effective amount of 0078. One approach to the study of cancer is genetic pro the drug may reduce the number of cancer cells; reduce the filing, an effort aimed at identifying perturbations in gene tumor size: inhibit (i.e., slow to some extent and preferably expression and/or mutation that lead to the malignant pheno stop) cancer cell infiltration into peripheral organs; inhibit type. These gene expression profiles and mutational status (i.e., slow to Some extent and preferably stop) tumor metasta provide valuable information about biological processes in sis; inhibit, to Some extent, tumor growth; and/or relieve to normal and disease cells. However, cancers differ widely in Some extent one or more of the symptoms associated with the their genetic signature, leading to difficulty in diagnosis and disorder. treatment, as well as in the development of effective thera 0073 For the recitation of numeric ranges herein, each peutics. Increasingly, gene mutations are being identified and intervening number there between with the same degree of exploited as tools for disease detection as well as for progno precision is explicitly contemplated. For example, for the sis and prospective assessment of therapeutic Success. range of 2-5, the numbers 3 and 4 are contemplated in addi 007.9 The inventors of the instant application hypoth tion to 2 and 5, and for the range 2.0-3.0, the number 2.0, 2.1, esized that gene expression profiling of brain metastasis 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 are explicitly would provide a more effective approach to cancer manage contemplated. As used herein, the term “about X or ment and/or treatment. The inventors have herein identified “approximately” X refers to +/-10% of the stated value of X. that altered expression of a panel of genes is predictive of 0074. Inherent difficulties in the diagnosis and treatment metastasis and likelihood of metastasis free survival (MFS). of cancer include among other things, the existence of many 0080. In particular, the present disclosure is directed, inter different Subgroups of cancer and the concomitant variation alia, to a method of predicting the likelihood that a patient inappropriate treatment strategies to maximize the likelihood with cancer will develop metastasis to the brain, bone and/or of positive patient outcome. Current methods of cancer treat lung. The method includes isolating a sample from the ment are relatively non-selective. Typically, Surgery is used to patient’s blood, primary tumor or metastatic tumor, then remove diseased tissue; radiotherapy is used to shrink Solid assaying the sample to determine the expression of cathepsin tumors; and chemotherapy is used to kill rapidly dividing S (CTSS) gene plus expression in at least one of genes cells. PSMB6, PSMD11, SLPI, PSMD13, and TIMP1 in said 0075 Often, diagnostic assays are directed by a medical sample. After this assaying step, the method includes (i) pre practitioner treating a patient, the diagnostic assays are per dicting that the patient with cancer will develop metastasis to formed by a technician who reports the results of the assay to the brain if, in addition to increased expression of cathepsin S the medical practitioner, and the medical practitioner uses the in the sample over control, expression of PSMB6 gene is values from the assays as criteria for diagnosing the patient. increased over control, (ii) predicting that the patient with Accordingly, the component steps of the method of the cancer will develop metastasis to the bone if in the sample present invention may be performed by more than one person. over control, expression of PSMD11 and SLPI gene is 0076 Prognosis may be a prediction of the likelihood that increased over control, and (iii) predicting that the patient a patient will survive for a particular period of time, or said with cancer will likely not develop metastasis to the bone if in prognosis is a prediction of how long a patient may live, or the the sample over control, expression of PSMD13 and TIMP1 prognosis is the likelihood that a patent will recover from a is increased. The primary cancer can be breast cancer. cathe disease or disorder. There are many ways that prognosis can psin Sand PSMB6 can be differentially expressed by both the be expressed. For example prognosis can be expressed in stroma and tumor in early and late stage brain metastasis. terms of complete remission rates (CR), overall survival (OS) PSMD11, SLPI, PSMD13, and TIMP1 can be differentially which is the amount of time from entry to death, disease-free expressed by both the stroma and tumor in early and late stage survival (DFS) which is the amount of time from CR to bone metastasis. relapse or death. In one embodiment, favorable likelihood of I0081 Methods of monitoring gene expression by moni survival, or overall survival, of the patient includes survival of toring RNA or protein levels are known in the art. RNA levels the patient for about eighteen months or more. can be measured by methods known to those of skill in the art US 2016/0138113 A1 May 19, 2016 including, for example, differential screening, Subtractive depending upon, for example, the component being admin hybridization, differential display, and microarrays. A variety istered, the patient and the condition of this patient, the mode of protocols for detecting and measuring the expression of of administration, and the type of cancer being treated. proteins, using either polyclonal or monoclonal antibodies specific for the proteins, are known in the art. Examples I0086. The present disclosure is also directed to a method include Western blotting, enzyme-linked immunosorbent of predicting the likelihood that a patient with cancer will assay (ELISA), radioimmunoassay (RIA), and fluorescence develop metastasis to the bone. The method comprises iso activated cell sorting (FACS). lating a genetic sample from the patient’s blood, primary 0082 Some methods require the use of probes and primers tumor or metastatic tumor, and Subsequently assaying the specific for an RNA transcript or other expression product of genetic sample to determine the expression in at least one of a gene of interest. A probe comprises an isolated nucleic acid genes PSMD11, TIMP1, PSMD13, and SLPI in said sample. attached to a detectable label or other reporter molecule. Having assayed for expression of these genes, the method Typical labels include radioactive isotopes, enzyme Sub includes (i) predicting that the patient with cancer will strates, co-factors, ligands, chemiluminescent or fluorescent develop metastasis to the bone if in the sample, expression of agents, haptens, and enzymes. Methods for labeling and guid PSMD11 and SLPI is increased over control, and/or (ii) pre ance in the choice of labels appropriate for various purposes dicting that the patient with cancer will likely not develop are discussed, e.g., in Sambrooketal. (In Molecular Cloning: metastasis to the bone if in the sample, expression of A Laboratory Manual, CSHL, New York, 1989) and Ausubel PSMD13 and TIMP1 is increased over control. et al. (In Current Protocols in Molecular Biology, John Wiley I0087 As well as brain and bone, metastasis to the lung is & Sons, New York, 1998). also of concern. For example, metastatic breast cancer, either 0.083 Primers are short nucleic acid molecules, for at the time of initial diagnosis or upon recurrence after an instance DNA oligonucleotides 10 nucleotides or more in initial treatment, commonly occurs in the bone, lung, brain or length. Longer DNA oligonucleotides may be about 15, 20, liver. Between 60% and 70% of women who die from breast 25, 30 or 50 nucleotides or more in length. Primers can be cancer have metastatic lung involvement, and in a significant annealed to a complementary target DNA strand by nucleic number of cases the lung is the only site of metastasis. The acid hybridization to form a hybrid between the primer and most common signs of lung metastases are: shortness of the target DNA strand, and then the primer extended along the breath and dry cough. In some cases, women will not expe target DNA strand by a DNA polymerase enzyme. Primer rience any symptoms; cancer will only be detected by chest pairs can be used for amplification of a nucleic acid sequence, X-ray or CT scan. Thus, the ability to identify early on those e.g., by the polymerase chain reaction (PCR) or other in vitro cancers that pose the greatest risk of lung metastasis overtime nucleic-acid amplification methods known in the art. These would provide an improved prognosis through the use of methods are within the skill of the ordinary artisan. increased monitoring. The present disclosure also teaches 0084 Diseases associated with bone metastasis include methods that relate using genes that are shown in FIG. 11E to cancers that spread from the primary tumor located in one part predict the likelihood of patients with primary or metastatic of the body to another. For example, an individual with pros tumors later developing lung metastasis. tate cancer may have a metastasis in their bone. Cells that metastasize are basically of the same kind as those in the I0088 Cathepsins are lysosomal cysteine proteases that original tumor, i.e.; if the cancer arose in the lung and metas belong to the papain superfamily. They are widely distributed tasized to the bone, the cancer cells growing in the bone are and differentially expressed among tissues. These enzymes lung cancer cells. Metastatic-associated diseases which may have a role in processes that involve proteolysis and turnover be treated by methods of the invention include, but are not of specific proteins and tissues. Cathepsins also participate to limited to, skin cancer, brain cancer, ovarian cancer, breast proenzyme activation and to antigen presentation by MHC cancer, cervical cancer, colorectal cancer, prostate cancer, class 2 proteins in antigen-presenting cells. The various mem liver cancer, lung cancer, stomach cancer, bone cancer, and bers of this family are differentially expressed, and some pancreatic cancer. forms of cathepsins are closely associated with monocytes, 0085. The drug combination of the invention may be used macrophages, and other cells of the immune system. The for the treatment of humans or animals with cancer, including secreted forms of several members of this family function in domestic, sport, laboratory, and farm animals. It is contem tissue remodelling through degradation of collagen, fibronec plated that the each component of the drug combination may tin, laminin, elastin, and other structural proteins and are beformulated into a pharmaceutical composition comprising implicated in the inflammatory response. an effective amount of the component and a pharmaceutically I0089 Cathepsin S, also known as CTSS, is a protein that in acceptable carrier. An effective amount of each component of humans is encoded by the CTSS gene. The term “cathepsin S' the drug combination may be administered to the patient in a has its general meaning in the art and refers to a secreted manner which, when combined with the other components of cysteine protease from the family of cathepsins. The term the drug combination, ultimately decreases the signs or symp may include naturally occurring "cathepsin S and variants toms of a disease associated with a bone metastasis. and modified forms thereof. The term may also refer to fusion Examples of signs and/or symptoms that may be monitored to proteins in which a domain from cathepsin S that retains the determine the effectiveness of the drug combination include, cathepsin S activity is fused, for example, to another polypep but are not limited to, PSA level, bone resorption, tumor size, tide (e.g., a polypeptide tag. Such as are conventional in the feelings of weakness, and pain perception. Beneficial effects art). The cathepsin S can be from any source, but typically is of the instant drug combination may, for instance, include a a mammalian (e.g., human and non-human primate) cathep 50%, 75% or 100% drop in PSA levels or a reduction in tumor sin S, particularly a human cathepsin S. An exemplary native size by 50%, 75% or 100%. The amount of each component cathepsin S amino acid sequence is provided in GenPept and the specific pharmaceutically acceptable carrier will vary database under accession number AAB22005 and an exem US 2016/0138113 A1 May 19, 2016
plary native nucleotide sequence encoding for cathepsin S is to a non-toxic Solid, semi-solid or liquid filler, diluent, encap provided in GenBank database under accession number NM Sulating material or formulation auxiliary of any type. OO4O79. 0097. One aspect of the present disclosure relates to meth 0090. The expression “inhibitor of cathepsin S should be ods and compositions (such as pharmaceutical compositions) understood broadly; it encompasses inhibitors of cathepsin S for treating and/or preventing metastatic cancer associated activity and inhibitors of cathepsin S expression. An “inhibi disorders, for example, brain metastasis. tor of expression” refers to a natural or synthetic compound 0098. The disclosure relates, interalia, to the use of inhibi that has a biological effect to inhibit or significantly reduce tors of cathepsin S activity for the treatment of brain metasta the expression of a gene. Consequently an “inhibitor of cathe sis and associated disorders. Particularly, the disclosure psin S expression” refers to a natural or synthetic compound relates to the use of selective inhibitors of cathepsin S activity that has a biological effect to inhibit or significantly reduce for the treatment and/or impairment of brain metastasis out the expression of the gene encoding for the cathepsin S gene. growth. In another embodiment, the disclosure relates to the 0091 Particularly, a “selective inhibitor of cathepsin S use of inhibitors of cathepsin S expression for the treatment of expression” refers to such compound which inhibits the brain metastasis. Particularly, the invention relates to the use cathepsin S expression more strongly than that of cathepsins of selective inhibitors of cathepsin S expression for the treat L or K expression in the sense that the inhibitor is at least 10 ment of brain metastasis. times, more preferably at least 100 times and most preferably 0099. In particular, the present disclosure is, in one at least 1000 times stronger inhibitor of the cathepsin S example, directed to a method of treating, preventing or man expression. aging metastasis of cancer cells from a primary tumor in a 0092 An “inhibitor of activity” has its general meaning in cancer patient to the patient's brain, bone and/or lung. The the art, and refers to a compound (natural or not) which has method comprises administering to the patient with cancer, the capability of reducing or Suppressing the activity of a for example breast cancer, an agent which inhibits cathepsin protein. It can be an antibody which binds the activity site of S. The agent can be a selective inhibitor of cathepsin S relative cathepsin S and inhibits its activity. Particularly, a “selective to a cysteine protease selected from cathepsins K, L, Hand B. inhibitor of cathepsin S activity” refers to such compound Alternatively, the agent can be a specific inhibitor of cathep which inhibits the cathepsin S activity more strongly than that sin S. The agent can be a peptide-based inhibitor of cathepsin of cathepsins L and Kactivity in the sense that the inhibitor is S, which is based upon a peptide sequence which comprises at least 10 times, more preferably at least 100 times and most 2-20 consecutive residues of a preferred invariant chain preferably at least 1000 times stronger inhibitor of the cathe cleavage site of cathepsin S. The agent may be administered psin S activity. As used herein, the term “subject' denotes a to the patient Suffering from cancer via intravenous injection, mammal. Such as a rodent, a feline, a canine, and a primate. intradermal injection, Subcutaneous injection, intramuscular Preferably, a subject according to the invention is a human. injection, intraperitoneal injection, anal Supposition, vaginal 0093. One aspect of the present disclosure a method of Supposition, oral ingestion or inhalation. The cathepsin S predicting metastasis of breast cancer to the brain, bone and/ inhibitor can be administered early on in the metastasis devel or lung of a patient Suffering from breast cancer. The method opment cascade. comprises obtaining a sample from the patient and analyzing 0100 Cathepsin Sinhibitors are known in the art and some it for increased expression of cathepsin S. The method are already approved or currently in clinical trials for indica includes (i) predicting the breast cancer patient has or is at risk tions such as systemic lupus erythematosus (SLE), psoriasis of developing metastasis to the brain if there is increased and irritable bowel syndrome. One example, VBY-129 (com expression of Cathepsin S gene in tumor cells early on in mercially available from Virobay, Inc.), is a potent, competi brain metastasis development, relative to control; and (ii) tive and reversible inhibitor of purified cathepsin S that is also predicting the breast cancer patient is not likely to develop highly selective against other human cathepsins (B. F. L. K metastasis to the bone and lung if there is increased expres and V). VBY-129 has potent activity in cellular assays and in sion of Cathepsin S gene in tumor cells early on in brain animal models of disease. metastasis development. Macrophages and/or primary tumor 0101 The peptide-based inhibitor of cathepsin S can be cells may be isolated as the sample. morpholinurea-leucine-homophenyl alanine-vinylsulfone 0094. In addition to increased expression of cathepsin S, phenyl (LHVS). The peptide-based inhibitor can be a pep the expression of one or more of the twenty one other genes tide-based vinylsulfone or a modified peptide-based vinylsul shown in FIG. 10C were increased with brain metastasis. fone. The peptide-based inhibitor can be selected from pep Further, the expression of one or more of the twenty five other tidyl aldehydes, nitriles, C.-ketocarbonyls, halomethyl genes shown in FIG. 10D were increased with bone metasta ketones, diazomethyl ketones, (acyloxy)-methyl ketones, sis. Further, the expression of one or more of the forty two vinyl Sulfones, ketomethylsulfonium salts, epoxides, and other genes shown in FIG. 10E were increased with lung N-peptidyl-O-acyl-hydroxylamines. The agent can be metastasis. selected from Asn-Leu-Vinylsulfone, Arg-Met-vinylsulfone, 0095. In one embodiment, in its broadest meaning, the Leu-Arg-Met-Vinylsulfone, Glu-Asn-Leu-Vinylsulfone, and term “preventing or “prevention” refers to preventing the Leu-Leu-Leu-Vinylsulfone. The agent can be selected from onset of or advancement of brain metastasis formation in a N-(carboxybenzyl)-Asn-Leu-vinylsulfone, N-(carboxyben Subject or Subject at risk of developing, for example, brain, Zyl)-Arg-Met-vinylsulfone, N-(carboxybenzyl)-Leu-Arg bone or lung metastasis. Met-vinylsulfone, N-(carboxybenzyl)-Glu-Asn-Leu-vinyl 0096 “Pharmaceutically” or “pharmaceutically accept sulfone, and N-(carboxybenzyl)-Leu-Leu-Leu-vinylsulfone. able” refers to molecularentities and compositions that do not 0102 One aspect of the present disclosure is a method of produce an adverse, allergic or other untoward reaction when treating, preventing or managing cancer cell metastasis in a administered to a mammal, especially a human, as appropri cancer patient. The method comprises extracting a sample ate. A pharmaceutically acceptable carrier or excipient refers from the primary tumor, metastatic tumor, or blood of the US 2016/0138113 A1 May 19, 2016
cancer patient, and then assaying the sample to determine the 0107. In still another embodiment, the inhibitor of cathe expression of cathepsin S and/or PSMB6 genes in said psin S activity is anaptamer. Aptamers are a class of molecule sample, and Subsequently administering a cathepsin Sinhibi that represents an alternative to antibodies in term of molecu tor if the expression of cathepsin S and/or PSMB6 genes is lar recognition. Aptamers are oligonucleotide or oligopeptide increased over control. sequences with the capacity to recognize virtually any class of 0103) In one embodiment, the inhibitor of cathepsin S target molecules with high affinity and specificity. Such activity may be an inhibitor of activity of this cathepsin, e.g. ligands may be isolated through Systematic Evolution of a small organic molecule. Several molecules have been Ligands by Exponential enrichment (SELEX) of a random described as inhibitors of cathepsin S activity. According to sequence library. The random sequence library is obtainable the invention, inhibitors of cathepsin S activity that could be by combinatorial chemical synthesis of DNA. In this library, used are described in Gauthier J Y et al., 2007. (The identifi each member is a linear oligomer, eventually chemically cation of potent, selective, and bioavailable cathepsin S modified, of a unique sequence. Peptide aptamers consists of inhibitors. Bioorganic & Medicinal Chemistry Letters 17 a conformationally constrained antibody variable region dis (2007) 4929-4933). played by a platform protein, such as E. coli Thioredoxin A 0104. Other examples of molecules that could be used are: that are selected from combinatorial libraries by two hybrid the Paecilopeptin, the dipeptide C.-keto-B-aldehyde or the methods. 4-Morpholineurea-Leu-HomoPhe-vinylsulphone (LHVS) or 0108. One aspect is directed to a kit for determining treat an antibody against cathepsin S described in the patent appli ment of a patient with brain metastasis. The kit comprises cation WO2007128987. These molecules can also derive means for detecting expression and/or activity of cathepsin S from the development of ligand-based and structure-based and/or PSMB6 genes at an early stage of brain metastasis. The pharmacophore models for noncovalent and covalent cathe kit also includes instructions for recommended treatment psin Sinhibitors (Markt et al.: Discovery of novel cathepsin S based on the presence of increased expression or activity in inhibitors by pharmacophore-based virtual high-throughput cathepsin S and/or PSMB6 genes. screening. JChem InfModel 48:1693-1705, 2008) or pyrrol opyrimidine-based inhibitors (Irie et al.: Discovery of selec Inhibitor of Cathepsin S Expression tive and nonpeptidic cathepsin S inhibitors. Bioorg Med 0109 Another aspect of the invention relates to selective Chem Lett 18:3959-3962, 2008). inhibitor of cathepsin S expression. Inhibitors of cathepsin S 0105. In another embodiment, the inhibitor of cathepsin S expression for use in the present invention may be based on activity is an antibody orantibody fragment that can partially anti-sense oligonucleotide constructs. Anti-sense oligonucle or completely blocks the cathepsin S enzymatic activity (i.e. otides, including anti-sense RNA molecules and anti-sense a partial or complete cathepsin S blocking antibody or anti DNA molecules, would act to directly block the translation of body fragment). In particular, the inhibitor of cathepsin S Cathepsin S mRNA by binding thereto and thus preventing activity may consist in an antibody directed against the cathe protein translation or increasing mRNA degradation, thus psin S, in such a way that said antibody blocks the activity of decreasing the level of Cathepsin S, and thus activity, in a cell. cathepsin S. Antibodies directed against the cathepsin S can For example, antisense oligonucleotides of at least about 15 be raised according to known methods by administering the bases and complementary to unique regions of the mRNA appropriate antigen or epitope to a host animal selected, e.g., transcript sequence encoding Cathepsin Scan be synthesized, from pigs, cows, horses, rabbits, goats, sheep, and mice, e.g., by conventional phosphodiester techniques and admin among others. Various adjuvants known in the art can be used istered by e.g., intravenous injection or infusion. Methods for to enhance antibody production. Although antibodies useful using antisense techniques for specifically inhibiting gene in practicing the invention can be polyclonal, monoclonal expression of genes whose sequence is known are well known antibodies are preferred. Monoclonal antibodies against in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365, cathepsin S can be prepared and isolated using any technique 354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). that provides for the production of antibody molecules by 0110 Small inhibitory RNAs (siRNAs) can also function continuous cell lines in culture. Techniques for production as inhibitors of Cathepsin S expression for use in the present and isolation include but are not limited to the hybridoma invention. Cathepsin S expression can be reduced by contact technique originally described by Kohler and Milstein; the ing a subject or cell with a small double stranded RNA human B-cell hybridoma technique and the EBV-hybridoma (dsRNA), or a vector or construct causing the production of a technique. Alternatively, techniques described for the pro small double stranded RNA, such that cathepsin S expression duction of single chain antibodies (e.g., U.S. Pat. No. 4,946, is specifically inhibited (i.e. RNA interference or RNAi). 778) can be adapted to produce anti-cathepsin S, single chain Methods for selecting an appropriate dsRNA or dsRNA-en antibodies. Cathepsin S inhibitors useful in practicing the coding vector are well known in the art for genes whose present invention also include anti-cathepsin S fragments sequence is known (see U.S. Pat. Nos. 6,573,099 and 6,506, including but not limited to F(ab')2 fragments, which can be 559) and International Patent Publication Nos. WO01/36646, generated by pepsin digestion of an intact antibody molecule, WO 99/32619, and WO 01/68836), shRNAs (short hairpin and Fab fragments, which can be generated by reducing the RNA) can also function as inhibitors of Cathepsin S expres disulfide bridges of the F(ab')2 fragments. Alternatively, Fab sion for use in the present invention. and/or schv expression libraries can be constructed to allow 0111 Antisense sequences to cathepsin S may readily be rapid identification of fragments having the desired specific chosen and produced by one of ordinary skill in the art on the ity to cathepsin S. basis of the known nucleic acid sequence of the cathepsin S 0106 Humanized and human anti-cathepsin Santibodies gene (see; e.g., GenBank Accession Nos. M86553, M90696, and antibody fragments thereof can also be prepared accord S39127; and Wiedersranders et at., J. Biol. Chem. 267: ing to known techniques. Methods for making antibodies, are 13708-13713 (1992)). In order to be sufficiently selective and well known in the art. potent for cathepsin Sinhibition, Such cathepsin S-antisense US 2016/0138113 A1 May 19, 2016 oligonucleotides should comprise at least 10 bases and, more advantageous for this because they do not have the same preferably, at least 15 bases. In one embodiment, the anti safety concerns as with many of the viral vectors. These sense oligonucleotides comprise 18-20 bases. plasmids, however, having a promoter compatible with the 0112 Ribozymes can also function as inhibitors of cathe host cell, can express a peptide from a gene operatively psin S expression for use in the present invention. Ribozymes encoded within the plasmid. Some commonly used plasmids are enzymatic RNA molecules capable of catalyzing the spe include pBR322, puC18, p OC19, pRC/CMV, SV40, and cific cleavage of RNA. The mechanism of ribozyme action pBlueScript, pSIREN. Other plasmids are well known to involves sequence specific hybridization of the ribozyme those of ordinary skill in the art. Additionally, plasmids may molecule to complementary target RNA, followed by endo be custom designed using restriction enzymes and ligation nucleo lyric cleavage. Engineered hairpin or hammerhead reactions to remove and add specific fragments of DNA. motif ribozyme molecules that specifically and efficiently Plasmids may be delivered by a variety of parental, mucosal catalyze endonucleolytic cleavage of cathepsin S mRNA and topical routes. For example, the DNA plasmid can be sequences are thereby useful within the scope of the present injected by intramuscular, intradermal, Subcutaneous, or invention. Specific ribozyme cleavage sites within any poten other routes. It may also be administered by intranasal sprays tial RNA target are initially identified by scanning the target or drops, rectal Suppository and orally. It may also be admin molecule for ribozyme cleavage sites, which typically istered into the epidermis or a mucosal Surface using a gene include the following sequences, GUA, GUU, and GUC. gun. The plasmids may be given in an aqueous Solution, dried Once identified, short RNA sequences of between about 15 onto gold particles or in association with another DNA deliv and 20 ribonucleotides corresponding to the region of the ery system including but not limited to liposomes, dendrim target gene containing the cleavage site can be evaluated for ers, cochleate and microencapsulation. predicted structural features, such as secondary structure, that 0116. In one embodiment, the disclosure relates to a phar can render the oligonucleotide sequence unsuitable. maceutical composition for treating and/or preventing brain 0113 Both antisense oligonucleotides and ribozymes use metastasis and/or associated disorders, said composition ful as inhibitors of cathepsin S expression can be prepared by comprising an inhibitor of cathepsin S expression and/or known methods. These include techniques for chemical Syn activity. In one embodiment, the inhibitor is a selective inhibi thesis such as, for example, by Solid phase phosphorothioate tor of cathepsin S expression and/or activity. chemical synthesis. Alternatively, anti-sense RNA molecules 0117 The inhibitor(s) of cathepsin S may be combined can be generated by in vitro or in vivo transcription of DNA with pharmaceutically acceptable excipients, and optionally sequences encoding the RNA molecule. Such DNA sustained-release matrices, such as biodegradable polymers, sequences can be incorporated into a wide variety of vectors to form therapeutic compositions. that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications 0118. In the pharmaceutical compositions of the present to the oligonucleotides of the invention can be introduced as invention for oral, Sublingual, Subcutaneous, intramuscular, a mean of increasing intracellular stability and half-life. Pos intravenous, transdermal, local or rectal administration, the sible modifications include but are not limited to the addition active principle, alone or in combination with another active of flanking sequences of ribonucleotides or deoxyribonucle principle, can be administered in a unit administration form, otides to the 5' and/or 3' ends of the molecule, or the use of as a mixture with conventional pharmaceutical Supports, to phosphorothioate or 2'-O-methyl rather than phosphodi animals and human beings. Suitable unit administration esterase linkages within the oligonucleotide backbone. forms comprise oral-route forms such as tablets, gel capsules, 0114 Antisense oligonucleotides, siRNAs, shRNAs and powders, granules and oral Suspensions or solutions, Sublin ribozymes of the invention may be delivered in vivo alone or gual and buccal administration forms, aerosols, implants, in association with a vector. In its broadest sense, a “vector' Subcutaneous, transdermal, topical, intraperitoneal, intra is any vehicle capable of facilitating the transfer of the anti muscular, intravenous, Subdermal, transdermal, intrathecal sense oligonucleotide, siRNA, shRNA or ribozyme nucleic and intranasal administration forms and rectal administration acid to the cells and preferably cells expressing Cathepsin S. forms. In general, the vectors useful in the invention include, but are 0119 The inhibitor of cathepsin S of the invention can be not limited to, plasmids, phagemids, viruses, other vehicles formulated into a composition in a neutral or salt form. Phar derived from viral or bacterial sources that have been manipu maceutically acceptable salts include the acid addition salts lated by the insertion or incorporation of the antisense oligo (formed with the free amino groups of the protein) and which nucleotide, siRNA, shRNA or ribozyme nucleic acid are formed with inorganic acids such as, for example, hydro sequences. Viral vectors are a preferred type of vector and chloric or phosphoric acids, or such organic acids as acetic, include, but are not limited to nucleic acid sequences from the oxalic, tartaric, mandelic, and the like. Salts formed with the following viruses: retrovirus, such as moloney murine leuke free carboxyl groups can also be derived from inorganic bases mia virus, harvey murine sarcoma virus, murine mammary Such as, for example, Sodium, potassium, ammonium, cal tumor virus, and rous sarcoma virus; adenovirus, adeno-as cium, or ferric hydroxides, and Such organic bases as isopro sociated virus; SV40-type viruses; polyomaviruses; Epstein pylamine, trimethylamine, histidine, procaine and the like. Barr viruses; papilloma viruses; herpes virus; vaccinia virus; 0.120. The carrier can also be a solvent or dispersion polio virus; and RNA virus such as a retrovirus. One can medium containing, for example, water, ethanol, polyol (for readily employ other vectors not named but known to the art. example, glycerol, propylene glycol, and liquid polyethylene 0115 One class of vectors includes plasmid vectors. Plas glycol, and the like), Suitable mixtures thereof, and Veg mid vectors have been extensively described in the art and are etables oils. The proper fluidity can be maintained, for well known to those of skill in the art. Recently, plasmid example, by the use of a coating, such as lecithin, by the vectors have been used as DNA vaccines for delivering anti maintenance of the required particle size in the case of dis gen-encoding genes to cells in vivo. They are particularly persion and by the use of Surfactants. US 2016/0138113 A1 May 19, 2016
0121 The inhibitor of cathepsin S of the invention may be tration of certain aspects and embodiments of the present formulated within a therapeutic mixture to comprise about invention, and are not intended to limit the invention in any 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or way. about 0.1 to 1.0 or even about 10 milligrams per dose or so. 0.126 Each of the applications and patents cited in this Multiple doses can also be administered. text, as well as each documentor reference cited in each of the 0122 One aspect of the present disclosure is directed to a applications and patents (“application cited documents'), and method of predicting the likelihood that a patient with cancer each of the PCT and foreign applications or patents corre will develop metastasis to the bone. The first step of the sponding to and/or paragraphing priority from any of these method includes isolating a genetic sample from the patients applications and patents, and each of the documents cited or blood, primary tumor or metastatic tumor. The sample is then referenced in each of the application cited documents, are assayed to determine the expression of ADAMDEC1 gene: hereby expressly incorporated herein by reference. More gen and based on the expression profile, once can predict (i) that erally, documents or references are cited in this text, either in the patient with cancer will develop metastasis to the brain a Reference List or in the text itself, and, each of these and/or lung if expression of ADAMDEC1 is increased over documents or references (“herein-cited references”), as well control, and/or (ii) that the patient with cancer will not as each document or reference cited in each of the herein develop metastasis to the bone if expression of ADAMDEC1 cited references (including any manufacturer's specifica is increased over control. tions, instructions, etc.), is hereby expressly incorporated 0123. Another aspect of the present disclosure is directed herein by reference. to a method of analyzing a cell expression profile for deter mining whether the cell is metastatic to the brain, bone or Differential Expression of Proteases and Protease Inhibitors lung. The method comprises extracting the cell, measuring an in Different Metastatic Microenvironments amount of cathepsin S, PSMB6, PSMD11, and SLPI nucleic I0127. In order to investigate tumor-stroma interactions in acid expression or polypeptide in the cell, and comparing the different metastatic environments we used a mouse model for amount of cathepsin S, PSMB6, PSMD11, and SLPI nucleic organ-specific experimental metastasis (FIG. 1a). In this acid expression or protein present in the cell to the amount of model, three different metastatic variants of the human breast cathepsin S, PSMB6, PSMD11, and SLPI nucleic acid cancer cell line MDA-MB-231 (Refs. 23-25) were injected expression or polypeptide in a sample isolated from normal, either intracardially or intravenously into immunocompro non-cancerous cells. Having done so, an amplified amount of mised mice, resulting in the development of brain, bone, or cathepsin S and PSMB6 nucleic acid expression or polypep lung metastases. While previous studies focused on tumor tide in the cell relative to the amount of cathepsin S and cell-specific expression changes identified by profiling each PSMB6 nucleic acid expression or polypeptide in the sample of these metastatic variant cell lines in culture', we have isolated from normal, non-cancerous cells indicates that can been able to additionally capture the stromal contribution by cer is likely to metastasize to the brain. On the other hand, an removing intact whole tumors at distinct stages of metastatic amplified amount of PSMD11 and SLPI nucleic acid expres seeding and outgrowth in different organs, and Subjecting sion or polypeptide in the cell relative to the amount of them to expression analyses (FIG. 1a, FIG. 8). PSMD11 and SLPI nucleic acid expression or polypeptide in I0128. An important technological advance that allowed us the sample isolated from normal, non-cancerous cells indi to simultaneously query tumor and stromal gene expression cates that cancer is likely to metastasize to the bone. The cell on the same platform is the “HuMu ProtIn custom array is, in one example, isolated from the patient’s blood, primary (Hu-Human, Mu-Murine, Prot=Protease and In=Inhibitor), tumor or metastatic tumor. In another example, the cell is which surveys the mRNA expression of proteases, their isolated from a primary breast tumor or a metastatic breast endogenous inhibitors and interacting partners. The tumor. uniqueness of this array is based on the species-specificity of 0.124. The disclosure is also directed to a method for pre the probe sets, with no cross-reactivity between the human paring a personalized genomics profile for a patient with and mouse genes. This platform thus allowed us to distin breast cancer. The method comprises extracting mononuclear guish between expression changes in the tumor (human) and cells or cancer cells from the primary tumor and Subjecting stromal (mouse) gene space in response to metastatic seeding them to gene expression analysis, and assaying the sample to and outgrowth (early- and late-stage metastases respectively, determine the expression of cathepsin S gene plus expression FIG. 8), with the goal of identifying tumor-stroma interac of at least one of genes PSMB6, PSMD11, and SLPI in said tions that modulate organ-specific metastasis. Each of the sample. The method also includes generating a report of the metastatic cell variants was transduced with a triple-fusion data obtained by the expression analysis, wherein the report TK-GFP-Luc imaging vector, enabling non-invasive biolu comprises a prediction of the likelihood of the patient being minescence imaging (BLI) as a read-out of metastatic burden, substantially free of metastasis to the brain if, in addition to as previously described. Early- and late-stage metastases in decreased expression of cathepsin S in the sample over con each organ site were harvested based on BLI output, as trol, expression of PSMB6 gene is also decreased over con described in further detail in the methods, and correspond to trol. The disclosure further comprises, in one example, pre micrometastatic and macrometastatic disease respectively. dicting that the patient with cancer will develop metastasis to I0129. Principal component analysis (PCA) was used to the bone if, in the sample over control, expression of evaluate the global trends in proteolytic network gene expres PSMD11 and SLPI gene is increased over control. sion across tissue and stage for both tumor cells and stromal Examples cells (FIG. 1b). Analysis of tumor cell-specific gene expres sion revealed pronounced changes between early- and late 0.125. The invention, having been generally described, stage metastases, across all three of the metastatic sites exam may be more readily understood by reference to the following ined. Meanwhile, stromal genes were differentially expressed examples, which are included merely for purposes of illus between early- and late-stage metastases in a tissue-depen US 2016/0138113 A1 May 19, 2016
dent manner. Specifically, the brain and bone stroma showed specificity of the HuMu arrays to separately profile stroma the most robust changes in gene expression as metastases and tumor-derived genes in a cross-species analysis. Weiden progressed from early- to late-stage. Across all tissues, there tified genes for which both human and mouse homologs were were few changes in gene expression between the normal significantly altered in each metastatic site (FIG. 2a, FIG. tissue (i.e. non-tumor burdened), and the stroma of early 11a, b). Cathepsin S showed a particularly intriguing expres metastases (brain, bone-0 genes, lung 3 genes, Table 8a). sion pattern: while tumor-derived cathepsin S was high in This could reflect the relatively low disease burden at the early brain metastases and decreased in late-stage metastases, early stages, resulting in a minimal impact on the organ as a stromal cathepsin S displayed the inverse pattern, with higher whole. Alternatively, this may indicate that expression expression in late-stage brain metastases compared to early changes in proteolytic genes in the stroma are not as impor stage. qPCR using species-specific probes for cathepsin S tant in the earliest stages of metastatic extravasation and confirmed these data in an independent sample set (FIG.2b). seeding, possibly due to a predominant role for tumor-Sup To distinguish between the cellular sources of cathepsin S, we plied proteases or non-protease factors in the stroma at this will refer to tumor/human CTSS in capitals, and stromal/ Stage. mouse CtSS in lower-case. 0130. Differential gene expression analyses revealed that I0133. Using a publicly available gene expression dataset many genes changed in tumor cells in the brain (242 genes), of locally advanced primary breast cancer with complete bone (241 genes) and lung (245 genes) between early- and clinical annotation (GSE 12276)23, we investigated whether late-stage metastases (FIG. 1c, Table 1b-d). By comparison, there were any associations between CTSS expression at the there were fewer stage-specific differentially expressed genes breast primary site and organ-specific metastasis-free Sur identified in the stroma of the brain (40 genes) and bone (44 vival (MFS). Patients were separated into three equal tertiles genes), and only one differentially expressed gene, haptoglo oflow, medium and high CTSS expression as described in the bin, in the lung stroma (FIG. 1d. Table 1e-g). In tumor cells, methods. Kaplan-Meier analysis was used to assess MFS for a Substantial proportion of differentially expressed genes brain, bone and lung metastasis. Interestingly, the high CTSS were shared across all three metastatic sites (176 genes, FIG. expression group was associated with decreased MFS only 9a), whereas few differentially expressed stromal genes were for the brain (FIG.2c). CTSS expression levels did not sig shared across >1 metastatic site (10 genes, FIG.9b). Rather, nificantly associate with either bone or lung MFS. This was there were multiple tissue-specific proteases and inhibitors in further evident in a complementary Cox proportional hazards the brain, bone and lung stroma (FIG.9c). Quantitative real time PCR (qPCR) confirmed the tissue-specific enrichment model analysis with a hazard ratio (HR) of 1.4 for brain MFS for representative candidates for each organ site in normal alone (95% confidence interval (C.I.) 1.05-1.89; P=0.0209) tissue, and early- and late-stage metastases (FIG. 9d). Repre (FIG.11c). sentative proteases and protease inhibitors that exhibited I0134. We used similar analyses to determine if other genes stage-specific gene expression changes between early and that were differentially expressed between early- and late late metastases were validated using immunostaining (FIG. stage metastases in the experimental model (FIG. 1c) were 10a-e) and qPCR (FIG. 10ft). In addition, we validated sev also associated with differences in patient survival (Table 2). eral of the genes identified in lung metastasis Xenografts We found that in addition to CTSS, 26 other genes were (Table 1a) in the immunocompetent MMTV-PyMT model of significantly associated with brain MFS. Of these, 23 genes breast-to-lung metastasis (FIG. 10k). were negatively associated with brain MFS. Only TPSG1. 0131 We also asked whether the expression changes in HNRNPC or SEPT2 expression was associated with stromal cells in organ-specific metastases are a general improved brain MFS (FIG. 11c, 1 Table 2a). 30 genes were response to tumor cell colonization of the respective tissue, or associated with bone MFS, of which 6 genes (MME, if the expression changes are specific to the metastatic cell SNRNP200, PSMB3, SLPI, PSMD10, and PSMD11) were variant used. In the models used herein, bone metastases negatively associated with bone MFS (FIG. 11d. Table 2b).59 occasionally develop in animals inoculated with the brain genes were associated with lung MFS, of which 45 genes metastatic (Br-M) variant, and conversely brain metastases were negatively associated with lung MFS (FIG. Ile, Table can be observed in mice inoculated with bone-metastatic 2c). Only one gene, SLPI, was found to be associated with (Bo-M) cells. This allowed us to compare stromal and tumor MFS in brain, bone and lung, where high expression corre gene expression in the matched (Br-M to brain, Bo-M to lated with poor patient prognosis (FIG. 11c-e). Although bone) and mismatched (Br-M to bone, Bo-M to brain) tumor cells underwent largely congruent changes in gene samples. Interestingly, for the genes tested, we found that expression from early- to late-stage metastases across the stromal gene expression changes depend on tumor-stroma three metastatic sites (FIG. 9a), only 20 of these genes were interactions that are specific to the metastatic tumor cell vari significantly associated with MFS at multiple sites, whereas ant (FIG. 10f g). By contrast, tumor gene expression in dif the majority of genes were associated with tissue-specific ferent metastatic variants responds to the same microenviron MFS (brain=11 genes, bone=24 genes, lung 40 genes) (FIG. ment in a similar manner, Suggesting an important effect of 11f. Table 2a-c). the stroma on the tumor gene expression program (FIG. 10h, I0135 CST7 in brain metastasis, together with CTSS and i). SERPINA3 in bone metastasis, were the only genes that Cathepsin S is Negatively Associated with Metastasis-Free showed the same stage-dependent and cell type-specific Survival in Patients with Brain Metastasis expression changes as CTSS in brain metastasis (FIG. 2a, 0132) While previous whole tumor analyses have been FIG. 11a). Given that we did not observe an association of useful for identifying genes associated with site-specific CTSS expression with patient bone MFS, and neither CST7 metastases, few studies have been able to identify genes that nor SERPINA3 expression associated with brain and bone are concordantly or discordantly expressed in tumor cells and MFS respectively (data not shown), we chose to further inves stroma. To address this, we took advantage of the species tigate the potential role of cathepsin S specifically in brain US 2016/0138113 A1 May 19, 2016 metastasis, a function not previously ascribed to this protease, whereas targeting either source separately had no effect. A or any cathepsin family member. separate cohort of mice for all four experimental groups was 0136. The patient expression data above was derived from aged until day 35 after tumor cell injection, which was whole tumor samples, thus precluding cell type-specific selected as the time point by which all mice in the control expression analyses. Wetherefore utilized an independent set group had developed brain metastases (FIG.3b). Ex vivo BLI of patient tissue samples of brain metastases, with matched analysis of the brain at this endpoint revealed a significant primary breast tumors in approximately half of the cases 64% decrease in signal output in the CTSS KD; Ctss KO (Table 3). Across all samples (breast cancer and brain group alone (FIG. 3c, d). Together, these results indicate that metastases), we found that the major cell types contributing to while there is a stage-dependency to cell type-specific cathe the tumor mass were cytokeratin (CK)+tumor cells (55-85%) psin S expression, contributions from both cellular sources and CD68+ macrophages (10-35%), with a minor fraction are required to regulate brain metastasis. representing CK-CD68- cells (FIG. 2d. e. FIG. 12a-d). We examined the cellular source of CTSS and found that the Cathepsin S Promotes Transmigration of the Blood-Brain highest level of CTSS staining (CTSS index) was in CD68+ Barrier by Metastatic Cells macrophages. CTSS was also expressed in CK+ tumor cells, I0139. To gain insights into the mechanisms underlying albeit at lower levels than in macrophages, in both primary impaired metastatic seeding and/or outgrowth specifically in tumors and matched brain metastases (FIG. 2d. f. FIG.12a, b, the CTSS KD; Ctss KO group, we next analyzed multiple e). Notably, CTSS expression in tumor cells was found in all tumorigenic processes in brain metastases at day 35. We molecular subtypes of breast cancer analyzed here (FIG. 2d, found that both the size and proliferation rate of tumors in f FIG. 12a, b, e, Table 3). CTSS KD; Ctss KO mice were significantly lower than any of the other groups (FIG. 3c-e), while we did not observe sig Combined Depletion of Cathepsin S in Tumor and Stromal nificant differences in the apoptosis rate at d35 between the Cells Reduces Experimental Brain Metastasis experimental groups (data not shown). In investigating these 0137 We next investigated the stromal cell source of Ctss phenotypes, it was evident that the small lesions which did in the experimental brain metastasis model. Seeding and out develop in the CTSS KD; Ctss KO mice were closely apposed growth of brain metastasis induced a pronounced stromal to the vasculature, with the majority of tumor cells being only response that was characterized by an accumulation of astro 1 cell diameter from the vessel, and there was a pronounced cytes and macrophages/microglia in metastatic lesions (FIG. reduction in growth at a distance from blood vessels (FIG.4a, 8d). Detection of cathepsin S using an antibody that recog b). Similarly, analysis of the area covered by GFP+ tumor nizes both mouse and human homologs, in combination with cells relative to the area covered by CD34+ blood vessels a range of cell-type specific markers, identified macrophages confirmed this significant reduction (FIG. 13e). This was not as the predominant stromal cell type expressing Ctss in brain a consequence of changes in blood vessel density, as there metastases and normal brain (FIG. 3a). We observed a were no differences across the experimental metastasis gradual increase of Ctss expression in Iba1 + macrophages groups (data not shown). Moreover, Ctss deletion did not alter from normal brain to early- and late-stage metastases. Inter blood vessel density or permeability in the normal brain of estingly, Ctss expression was highly induced in Iba1 + mac non-tumor bearing animals (FIG. 13f, g). These results are rophages that were localized in close proximity to metastases. Suggestive of either a potential defect in seeding of single CTSS expression was also detectable in tumor cells, though at brain metastatic cells in the earliest stages, or a subsequent lower levels than in macrophages, mirroring the patient impairment in colonization, or perhaps insufficiencies in both analyses. At late stages, CTSS expression was undetectable in processes. the majority of the tumor cells. We found a similar expression I0140. To further investigate these possibilities, we pattern in an immunocompetent brain metastasis model (FIG. assessed metastatic seeding in the experimental metastasis 101). These data confirm the stage- and cell type-dependent model across the four experimental groups. We examined the expression changes at the protein level as predicted by the earliest steps of brain metastatic cell homing and survival29. HuMu array. specifically the first 48 h. We found that 24 h after CTSS KD 0138 Given the reciprocal, cell type-specific expression tumor cell injection, there was a reduction in BLI signal in pattern of cathepsin S, we next sought to investigate if the both WT and Ctss KO mice (FIG. 5a, b). The experimental tumor and stromal sources play important, perhaps comple group in which CTSS KD cells were injected into WT mice mentary roles in the seeding and outgrowth of experimental showed a BLI signal close to the control group after 48 h, brain metastases. To address this, we performed short hairpin while there was a progressive decrease in BLI signal in the (sh)-RNA-mediated CTSS knockdown (KD) in the brain CTSS KD; Ctss KO group (FIG.5a, b). Similarly, analysis of metastatic (Br-M) cells, achieving a 90% reduction of CTSS the proportion of viable tumor cells still within the blood expression at both the mRNA and protein level, and a corre Vessel lumen (intravascular), in the process of extravasating, sponding reduction in secreted CTSS protein (FIG. 13a-c). or fully extravascular, revealed significant differences in the There was no effect of CTSS knockdown on tumor cell pro CTSS KD; WT group at 24 h, and in the CTSS KD; Ctss KO liferation in culture (FIG. 13d). After backcrossing Ctss group at both 24 h and 48 h (FIG. 5c). knockout (KO) mice28 into the Athy/nu background, we 0141 Given that there was an initial reduction in tumor generated four experimental groups (shown in FIG. 3b) to cell extravasation in the CTSS KD; Ctss WT group (FIG.5a, analyze the effects of targeting tumor or stromal cathepsin S c), although the incidence of detectable brain metastasis was alone, or in combination, compared to the control group. ultimately not affected (FIG. 3b), we assessed subsequent Interestingly, only the combined removal of tumor and stro metastatic colony outgrowth. While there was an initial trend mal cathepsin S significantly reduced brain metastasis inci towards delayed growth in the CTSS KD; Ctss WT cohort, dence, as monitored by BLI output (FIG. 3b, Control (Ctrl): brain tumors ultimately grew to the same extent as the con Ctss wild-type (WT) vs. CTSS KD; Ctss KO, P<0.001), trols (FIG. 13h). In contrast, the kinetics of tumor growth in US 2016/0138113 A1 May 19, 2016
the CTSS KD; Ctss KO group did not recover over the same (FIG. 6a). We next examined the mRNA expression levels of time course (FIG. 13h). These results suggest that tumor- and each of these junctional components in both endothelial cell stromal-derived cathepsin S show some functional redun types (HBMECs and HUVECS) that were used for the invitro dancy during seeding and outgrowth and that the impact of BBB assay. Interestingly, we found that JAM-B levels were each cellular source is most likely regulated by differential significantly higher in HBMECs compared to HUVECs. expression levels at distinct stages. Additionally, tumor cell JAM-B was the only candidate substrate that displayed this derived-CTSS may be important for the initial steps of blood differential expression; the other junctional proteins were brain barrier (BBB) transmigration and extravasation into the generally expressed at higher levels in HUVECs (FIG. 6b). brain, whereas stromal-Supplied CtSS is Subsequently Staining of several junctional proteins revealed tissue-spe involved in Supporting tumor cell Survival to Successfully cific expression for Jam-B, which was detectable only in form brain micrometastases, and only their combined deple brain, and not bone or lung (FIG. 6c). Occludin and Cldn3 tion impairs the entire cascade of metastatic seeding and showed a somewhat broader expression pattern (FIG. 6c). outgrowth. Interestingly, a similar finding was recently The tissue-specific enrichment of genes encoding junctional reported in a colorectal carcinoma model, where depletion of proteins was confirmed by querying publicly available both tumor and stromal sources of cathepsin S was also datasets (FIG.14c). required to slow tumor growth. 0144. As the effects of CTSS depletion or inhibition on Br-M transmigration were only observed when HBMECs Cathepsin S Promotes BBB Transmigration Via Junctional were used in the BBB assay, and given the organ-specificity Protein Cleavage of Jam-B expression (FIG. 6c), we reasoned that JAM-B 0142. The BBB is a selective barrier between the systemic might be the most relevant substrate in this assay. We next circulation and the brain, which is formed by specialized aimed to identify the putative cleavage location for CTSS in endothelial cells, pericytes and astrocytes31. While the BBB JAM-B. We compared the fragment sizes of each cleavage restricts the entry of most macromolecules, it is not an impen product that was detectable with JAM-A, -B, or -C specific etrable barrier in transmigration of metastasizing cancer cells antibodies to fragments that contain the immunoglobulin (Ig) into the brain. We therefore examined the potential role of G1 domain, which is linked to recombinant JAM proteins tumor cell-supplied CTSS in breaching the BBB, by using an (FIG.14d). The molecular weight of the JAM cleavage prod in vitro BBB assay32. We performed either genetic or phar ucts and pH dependence of JAM processing by CTSS sug macological depletion of CTSS in Br-M cells via shRNA gests that all 3 family members share a similar but not fully mediated knockdown, or a cathepsin S-specific inhibitor conserved CTSS cleavage site that is localized close to the VBY-999 respectively, which does not affect viability of transmembrane domain. Alignment of the amino acid Br-M cells (FIG. 14a). Inhibition or knockdown of CTSS did sequence of the JAM family members identified the sequence not affect the ability of Br-M cells to cross a BBB formed by indicated in FIG. 14e as the putative cleavage site for CTSS, human umbilical endothelial vein cells (HUVECs) and astro which contains a sequence consistent with specificity prefer cytes (FIG. 14b). By contrast, when human brain microvas ences for CTSS that were previously identified in biochemi cular endothelial cells (HBMECs) were used instead of cal studies''. Cleavage in this region of the JAM proteins HUVECs, there was a significant reduction in Br-M cells likely leads to shedding of the JAM extracellular domain, crossing the BBB, and this was further impaired by 55-65% thereby disrupting cell-cell adhesion. We performed cell via genetic or pharmacological depletion of CTSS respec based cleavage assays as illustrated in FIG. 6d to test if tumor tively (FIG. 5d). Cell layers that were formed by HBMECs cell-secreted CTSS mediates shedding of JAM-B from the without the addition of astrocytes also significantly decreased HBMEC cell surface. Indeed, incubation of HBMECs with tumor cell-conditioned media (CM) led to a CTSS-mediated the transmigration capability of CTSS KD Br-M cells (FIG. accumulation of JAM-B in HBMEC CM after 2–4 h. The 14b), whereas transmigration of Br-M cells across HUVECs effect was decreased by the addition of the cathepsin S-spe or astrocytes alone was not altered by CTSS depletion (FIG. cific inhibitor VBY-999 (FIG. 6e, FIG. 14f). These results are 14b). consistent with the impairment of BBB transmigration in 0143 Tight junctions and adherens junctions between vitro and in vivo when CTSS is targeted, as CTSS-mediated adjacent cells are critical for maintaining BBB integrity, and shedding of the JAM-B extracellular domain would be are composed of different proteins including junctional adhe expected to disrupt the integrity of tight junctions thereby sion molecules (JAMs), occludin, claudins and cadherins31, 33, 34. Therefore, we investigated whether any of these pro allowing tumor cells to breach the BBB. teins represented potential CTSS substrates. We first per Cathepsin S Inhibition Reduces Experimental Brain formed biochemical cleavage assays using recombinant Metastasis Formation CTSS and recombinant proteins for each of the potential Substrates, under similar conditions to those we previously 0145 Given our identification of cathepsin S as an impor reported for the identification of E-cadherin cleavage by tant regulator of brain metastasis in experimental models and CTSS6. CTSS efficiently cleaved the three JAM family mem the negative association with patient Survival, we examined bers JAM-A, -B and -C at pH 4.5, the acidic pH of the whether its pharmacological inhibition is sufficient to reduce lysosome, and maintained robust cleavage of JAM-B specifi metastatic seeding and colonization in a preclinical preven cally at pH 6.0, the acidified pericellular pH measured in solid tion trial (FIG. 7a). Mice were treated with VBY-999 for 2 tumors35. Importantly, cathepsin S retains activity even at days to inhibit cathepsin S activity prior to tumor cell inocu neutral pH36. JAM cleavage was inhibited by the cathepsin lation, and were then continuously treated with VBY-999 S-specific inhibitor VBY-999 in all cases (FIG. 6a). Occludin until the trial endpoint of 35 days post-tumor cell inoculation. and Claudin (CLDN)-3 were also cleaved by CTSS, whereas Pharmacokinetic analysis showed that VBY-999 levels in the CLDN5 and the adherens junction proteins CD31 and CDH5 plasma were significantly above the required concentration (VE-cadherin) were unaffected by incubation with CTSS for target inhibition at the time of tumor cell inoculation, and US 2016/0138113 A1 May 19, 2016
confirmed that VBY-999 efficiently crosses the BBB with static cells (Bo-M) were inoculated into the left cardiac ven stable concentrations in the brain throughout the duration of tricle of 6-8 week old female Athymic/nude mice. For lung the trial (FIG. 7b). Interestingly, we found a significant metastasis generation, 1x10 lung-metastatic cells (Lu-M) 65-77% reduction in BLI signal at different time points dur were injected into the lateral tail vein of 6-8 week old female ing the trial, and at the trial endpoint of 35 days (FIG. 7c, d. NOD/SCID mice. P<0.05). 0151. For brain metastasis generation in immunocompe 0146 Initiation of VBY-999 treatmentinfully established, tent mice, 1x10 PyMT-BrM cells were inoculated into the end-stage brain metastases did not result in a significant dif left cardiac ventricle of 6-8 week old female FVB/n mice. ference in tumor burden (FIG. 13i,j), indicating that targeting Early and late metastases were defined by their biolumines this enzyme is most critical in seeding and early outgrowth. cence intensity (BLI) at the time of tissue harvest for samples We also investigated the organ specificity of cathepsin S used in the microarray analysis and the independent sample inhibition by assessing bone metastasis in a prevention trial set used for validation. Brain metastases that had a BLI output setting, using two different approaches. First, as bone and between 4.3x106 to 4.2x107 photons/sec were classified as spine metastases can develop in the brain metastasis model, early-stage metastases and were collected between 3-4 weeks we assessed whether there was an effect on these lesions after tumor cell inoculation. Late-stage brain metastases had following VBY-999 treatment. There was no significant dif a BLI output between 1.6x10 to 6.4x10 photons/sec and ference between the treatment groups, which was further were collected between 5-8 weeks after tumor cell inocula Supported by the finding that genetic depletion of cathepsin S tion. Histological and morphometric analyses of these differ also had no effect on bone metastasis formation (FIG. 13 k, l). ent stages showed that early-stage brain metastases are com Similarly, VBY-999 treatment of the bone metastasis model prised of clusters of ~50-200 cells, and can be considered specifically in a prevention trial showed no change in BLI similar to micrometastases, and late-stage metastases con output or development of osteolytic metastases (FIG. 7e, f. sist of clusters of ~5,000-15,000 cells, corresponding to FIG. 13 m). These data are consistent with our finding that macrometastases. Representative images of the different CTSS expression levels in patients only correlated with brain stages are shown in FIG. 8d. Early-stage bone metastases MFS, and not bone MFS. In sum, cathepsin S inhibition is were defined by a BLI intensity that ranged between 6.3x10 efficient in Substantially and specifically reducing brain to 1.1x10 photons/sec and were harvested 3 weeks after metastasis if cathepsin S activity is blocked throughout the tumor cell inoculation. Late-stage bone metastases showed a course of experimental brain metastasis. minimal BLI intensity of 8x10 photons/sec and a maximal BLI intensity of 2.5x10° photons/sec and were harvested 5 Mice weeks after tumor cell inoculation. Histological and morpho 0147 All animal studies were approved by the Institu metric analyses of bone metastases showed that early-stage tional Animal Care and Use Committee of Memorial Sloan lesions are comprised of clusters of -50-200 cells, and late Kettering Cancer Center. Athymic/nude mice were purchased stage metastatic clusters consist of -2.000-10,000 cells. Rep from NCI Frederick or bred within the MSKCC animal facil resentative images of the different stages are shown in FIG. ity. The cathepsin S knockout mouse line (Ctss KO) was 8e. The generation of mismatched samples (Br-Min bone or generated as described previously' and backcrossed for 6 Bo-M in brain) followed the same criteria for early- and generations to the Athymic/nude background. NOD/SCID late-stage metastasis. Early-stage lung metastases were har mice were purchased from Charles River Laboratories. vested 48 h after tumor cell inoculation. The BLI intensity at MMTV-PyMT immunocompetent transgenic mice (FVB/ this time point ranged between 2.1x10° to 1.7x107 photons/ n) were bred within the MSKCC animal facility. Sec. Late-stage lung metastases were harvested 5 weeks after tumor cell inoculation with an average BLI intensity between Cell Lines 8.1x10 to 3x10° photons/sec. Histological and morphomet 0148 Brain- (Br-M), bone- (Bo-M) and lung- (Lu-M) ric analyses of lung metastases showed that early-stage metastatic variants of the human breast cancer cell line MDA lesions are comprised of cells diffusely present throughout MB-231 (denoted parental) were generated as previously the lung (~2,000-4,000 cells per sectional plane, per entire described'' and labeled with the triple imaging vector lung), and late-stage metastatic clusters consist of ~1,000-5, (TK-GFP-Luc; TGL)7 to allow for non-invasive in vivo 000 cells. Representative images of the different stages are imaging of tumor growth overtime. The MDA-MB-231 vari shown in FIG. 8f. Late-stage lung metastases from the spon ants were cultured in DMEM+10% FBS. Mouse Br-M vari taneous MMTV-PyMT breast-to-lung metastasis model were ants were derived from the TS1 cell line that was previously harvested from 14 week-old female PyMT mice. isolated from MMTV-PyMT mammary tumors. These are 0152 For tissue isolation, mice were lethally anesthetized denoted PyMT-BrM cells. with 10 mg/ml ketamine/1 mg/mlxylazine and retro-orbitally 0149 Human Umbilical Vein Endothelial Cells (HUVEC) injected with 15 mg/ml luciferin. Mice were then intracar were purchased from the ATCC. Human Brain Microvascular dially perfused with PBS. Tumor-burdened tissue was iden Endothelial Cells (HBMEC) and Human Astrocytes (HA) tified by the presence of BLI signal for brain and bone were purchased from Sciencell. HUVEC and HBMEC were metastases. For lung metastases, part of the left lung lobe was cultured on gelatin coated cell culture dishes, and HA on collected. Snap frozen samples were collected for RNA and poly-L-lysine coated cell culture dishes in endothelial cell protein isolation and tissues were fixed in 4% paraformalde media (ECM, Sciencell)+10% FBS supplemented with hyde (PFA) for histology. endothelial cell growth factors (ECGF). Microarray Analysis Generation of Brain, Bone and Lung Metastases 0153. For microarray analysis, all samples were prepared 0150. For brain and bone metastases in xenografted mice, and processed by the Genomics Core Facility at MSKCC. 1x10" brain-metastatic cells (Br-M) or 1x10 bone-meta RNA was isolated using Trizol (Invitrogen) and the quality US 2016/0138113 A1 May 19, 2016
was assessed by running on an Agilent Bioanalyzer. Total Generation of CTSS Knock-Down Lines RNA was reverse transcribed and labeled using the Genechip 3' IVT Express Kit (Affymetrix). The resulting cRNA was 0158. Five shRNA sequences targeting CTSS were hybridized to HuMu Prot/In chips (Affymetrix). All bioinfor obtained from the RNAi Codex and RNAi Consortium. matics analyses were completed in Rusing the Bioconductor shRNA sequences were inserted into the targeting hairpin Suite of packages. The affy package was used for robust sequence for the pRetroSuper vector. Correct insertion into multi-array average normalization followed by quantile nor the vector was verified by digestion and sequencing of the malization. Mouse and human samples and probes were nor vector. Plasmids with the correct shRNA targeting sequence malized separately. With the exception of the cross-species were transfected into H29 viral packaging cells. Viral par ticles were concentrated from the H29 cell supernatant, added scatterplots, all Subsequent bioinformatics analyses regarded to the target cells in the presence of polybrene and cells were the tumor and stroma separately. selected with puromycin. One of the four shRNAs (CTSS 0154 The limma package was used to identify differen shRNA: GATAAAGTTTGCTAAGTAA TTACTTAG tially expressed genes across tissue and metastatic stage for CAAACTTTATC) was used for subsequent experiments to both tumor and stroma. Differential expression was consid target CTSS with 90%. KD efficiency. A non-targeting shRNA ered significant at a fold change of t2 with a false discovery (CGCCATAAATATAACTTTA TAAAGTTATATTTATG rate of 10%. Tissue-specific genes were identified by the GCG was used as control. intersection of pairwise comparisons: e.g. lung stroma-spe cific genes were identified by the intersection of genes sig Targeting Tumor- and Stroma-Derived CTSS InVivo nificantly enriched in lung VS. bone and genes significantly enriched in lung VS. brain. Stage-specific genes were identi 0159 1x10 Br-M cells (Br-M CTSS KD or Br-M Ctrl) fied in a tissue-specific manner comparing early- and late were inoculated into the left ventricle of 6-8 week old female stage metastases. The microarray data is deposited at NCBI Athymic/nude or Ctss KO Athymic/nude mice. Metastases formation was monitored once per week by bioluminescence GEO under the accession number GSE47930. imaging using a Xenogen IVIS-200 Optical In Vivo Imaging 0155 Principal component analysis (PCA) was com System to determine metastasis incidence in the four experi pleted using the covariance matrix in the princomp package mental groups shown in the table in FIG. 3b. In addition, in R. The first two components are plotted in FIG. 1b. numberical values of the increase in BLI intensity present the Homologs for mouse and human genes were identified using kinetics of tumor progression (FIG. 13h). An independent the HomoloGene Database through the NCBI (url www.ncbi. cohort of mice was injected with Br-M cells as described nlm.nih.gov/homologene). Homolog pairs were plotted with above and sacrificed at day 35 after tumor cell inoculation for mouse/stroma tissue-specific, early VS. late, fold change on Subsequent analysis of proliferation, apoptosis, angiogenesis, the X axis, and human/tumor tissue-specific, early vs. late, and metastatic outgrowth. fold change on the y axis. 0160 For in vivo extravasation experiments, Athymic/ nude or Ctss KO Athymic/nude mice were inoculated with External Datasets and Survival Analysis 5x10 Br-MCtrl or Br-MCTSS KD cells. BLI intensity was monitored Oh, 24 hand 48 h after tumor cell inoculation and 0156 For gene expression analysis of mouse endothelial the BLI intensity was plotted relative to the BLI intensity cells, raw data from GSE4706737 was imported into R and immediately after tumor cell inoculation (Oh time point). normalized as above. For patient analysis, normalized gene expression data was downloaded from the GEO (GSE 12276). Identification of Cathepsin S Inhibitor VBY-999 Each gene was mean centered and scaled by Standard devia (0161 VBY-999 was provided by Virobay Inc., Menlo tion. Patients were split into tertiles (lower 33%, middle 33%, Park, Calif. and is part of an extensive structure-based drug upper 33%) of CTSS gene expression for Kaplan-Meier sur discovery program. VBY-999 is a covalent reversible inhibi vival analysis. The scaled, continuous CTSS gene expression tor with an electrophilic nitrile warhead. The detailed chemi was used for Hazard Ratio (HR) calculation. Similar analyses cal synthesis and structure of compounds in the structural were completed for genes in FIG. 11c-e. Survival analysis series including VBY-999 can be found in issued U.S. Pat. was completed using the survival package in R. Hazard No. 7,547,701. Recombinant purified human and mouse ratios were determined utilizing the coxph function from the cathepsin S were used to assess potency of VBY-999 and survival package. Nominal P values are reported for HR determine inhibition constants. Activity on the peptide sub significance in Table 2 with a significance cutoff of 0.05 used strate Z-Leu-Arg-AMC was determined in vitro by measur to identify genes significantly associated with metastasis-free ing hydrolysis of the Substrate with spectrofluorimetric quan Survival. P values were generated using the log-rank statistic titation of AMC. The VBY-999 inhibitor was preincubated for Kaplan-Meier analysis and Wald’s test for the Hazard with cathepsin Sfor 15 minat room temperature (25°C.) after Ratio analysis. which the substrate was added to initiate the 30 min reaction. Assay incubation buffer included 25 mM CH3COONa, pH Clinical Samples 4.5, 2.5 mM DTT, and 0.05 M. NaCl. Appropriate reaction conditions and peptide Substrates for other cysteine and 0157. The specimen of primary breast tumors and brain serine proteases were utilized to screen for selectivity of metastases used in this study were obtained at MSKCC, Mas VBY-999 for cathepsin S. VBY-999 has an inhibition con sachusetts General Hospital (MGH), Brigham and Women's stant Ki(app)=290 pM on the purified human cathepsin S Hospital (BWH) and Dana Farber Cancer Institute according enzyme, and >3000-fold selectivity versus the related cathe to protocols approved by the human Subjects institutional psins K, L, B, and F. Potency on the closely related cathepsins review boards of MSKCC, DFCI, MGH, and BWH. Infor K, L, and F was Ki(app)>3 uM, with potency on cathepsin B mation about the clinical samples can be found in Table 3. Ki(app)=700 nM. Potency on mouse cathepsin S enzyme was US 2016/0138113 A1 May 19, 2016 20 verified on mouse cathepsin Spurified enzyme. VBY-999 has through 0.22 Lum filters to remove cellular debris. For western an inhibition constant Ki(app)=690 pM on mouse cathepsin blotting, CM was concentrated by centrifugation in Centrifu S. No measurable inhibition was detected for any other cys gal Filter Units (Millipore). For protein isolation from cells in teine, serine or aspartyl proteases tested. monolayer, cells were harvested by Scraping and lysed in RIPA lysis buffer (Pierce) with 1x complete Mini protease VBY-999 Inhibitor Preclinical Trial inhibitor cocktail (Roche). For protein isolation from tissue, snap frozen tissue was homogenized in RIPA lysis buffer (0162 For administration to mice, the VBY-999 inhibitor (Pierce) with 1x complete Mini protease inhibitor cocktail was formulated in a nanoparticle-based suspension formula (Roche) followed by Dounce homogenization. Protein was tion and further diluted in 5% dextrose in water (D5W) at a quantified using the BCA assay (Pierce). Protein lysates were concentration of 10 mg/ml. Subcutaneous dosing of VBY loaded onto SDS-PAGEgels and transferred to PVDF mem 999 provided a dosing formulation and route that allows high branes for immunoblotting. Membranes were probed with and Sustained plasma concentrations of the drug to be antibodies as indicated in Table 5 and detected using the achieved, which was confirmed using a bioanalytical LC-MS/ appropriate HRP-conjugated secondary antibodies using MS method after 2 and 7 days of treatment (FIG. 7b). This chemoluminescence detection (Millipore). Bands from west results in full inhibition of the enzyme target for the duration ern blots were quantified in the dynamic range using the Gel of the trial, following once-daily dosing. In order to determine analysis module in Image.J Software. if VBY-999 had sufficient penetration of the CNS to be avail able for cathepsin S inhibition in the brain and at the blood Generation of Serpina3n Antibody brain barrier site, and to confirm that concentrations in the brain remain stable throughout the duration of the trial, VBY 0.165 Peptides targeting murine Serpina3n were deter 999 concentration was determined at day 2, day 7, and day 37 mined via alignment of the protein sequence for serpina3in after treatment start by LC-MS/MS (FIG. 7b). These data against mouse Serpin a3 family members, Serpina3b, c, f, g, indicate that VBY-999 levels in the plasma were significantly k, and m, as well as human SERPINA3. From this alignment, above the required concentration for target inhibition at the divergent regions were located and peptides were chosen that time of tumor cell inoculation and that VBY-999 levels in the corresponded to regions 373-396 (a3n-no.1), 225-248 (a3n brain remainstable throughout the 37-day treatment schedule no2), and 398-418 (a3n-no3) of Serpina3n. 10-14 mg of each at a level sufficiently greater than the enzyme inhibition con peptide was synthesized by the Pocono Rabbit Farm and stant, and are thus expected to effectively inhibit cathepsin S Laboratory, with 2 mg of each peptide conjugated to KLH and activity. For the prevention trials, mice were randomly 2 mg of each peptide conjugated to BSA. The KLH-conju assigned into vehicle and VBY-999 treatment groups and gated peptides were used to generate an immune response in treatment was commenced two days before tumor cell inocu Armenian hamsters and BALB/c mice by the Monoclonal lation (d=-2). Mice were dosed with 100 mg/kg VBY-999 or Antibody Core Facility at MSKCC. Serum from hamsters and vehicle (D5W) by subcutaneous injection once daily. At day mice was tested via ELISA using the BSA-conjugated pep 0, Athymic/nude mice were inoculated with 1x104 Br-MCtrl tides in Nunc Maxisorp ELISA plates (protocol provided by cells or 1x105 Bo-M Ctrl cells. Metastases formation was the MAb core). The best responding hamster to all three monitored every fifth day by bioluminescence imaging using peptides was used for fusion, and positive colonies were a Xenogen IVIS-200 Optical InVivo Imaging System during screened by ELISA and for response to each peptide. Ten the trial period from day 0 to day 35 after tumor cell inocu positive colonies were saved for each peptide. Clones were lation. For the Bo-M trial, mice were subjected to X-ray also screened by immunohistochemistry and immunofluores analysis at d35 after tumor cell inoculation using a SPECT cence for ability to recognize murine Serpina3n in mouse CT scanner (X-SPECT). For the regression trial, mice were tissue. One colony (13H5, which responded to peptide a3n stratified into vehicle and VBY-999 treatment groups at d27 no1) was selected for Subcloning. after tumor cell inoculation to achieve equal average BLI intensity at the time of treatment start at d28. Mice were dosed Immunocytochemistry daily with either vehicle or VBY-999 (100 mg/kg) for 7 days and metastasis growth was monitored by BLI imaging at d32 0166 For immunocytochemistry, cells were cultured on and d35. glass coverslips and fixed in 4% paraformaldehyde in 0.1M RNAIsolation, cDNA Synthesis and Quantitative Real-Time phosphate buffer for 20 min at room temperature. Cells were PCR permeabilized in PBS with 0.25% Triton X-100 for 10 min. Cells were blocked in 0.5% PNB (phosphate-NaCl) in PBS (0163. RNA was isolated with Trizol, DNase treated, and for at least 1 hour at room temperature, followed by incuba 0.2 g of RNA was used for cDNA synthesis. Details about tion in goat anti-human CTSS primary antibody diluted 1:100 the Taqman assays can be found in Table 4. All species in 0.25% PNB overnight at 4° C. Cells were then washed in specific Taqman assays were chosen based on their location in PBS and incubated with the donkey anti-goat Alexa568 sec the mRNA sequence that allows for maximal discrimination ondary antibody (Molecular Probes) at a dilution 1:500 in between mouse and human transcripts. For each Taqman 0.25% PNB for 1 hour at room temperature. After washing in assay, species specificity was tested by qPCR using mouse or PBS, cells were counterstained with DAPI (5 mg/ml stock human samples as controls. diluted 1:5,000 in PBS) for 5 minutes prior to mounting with ProLong Gold Antifade mounting media (Invitrogen). Collection of Conditioned Media, Protein Isolation and Western Blotting. 0.167 Paraffin-embedded sections were processed using a Ventana automated Staining device. The automated deparaf 0164 Conditioned media (CM) from Br-M cell lines was finization/rehydration, citrate buffer-based antigen retrieval, generated by incubating confluent cell layers in serum-free and blocking of unspecific protein binding and endogenous DMEM media for 24 hours. Collected (CM) was passed peroxidase was followed by incubation with mouse anti-hu US 2016/0138113 A1 May 19, 2016 man CD68 (Dako) primary antibody and goat anti-human automatically acquired using TissueCuest Software (Tissue CTSS (R&D Systems) or mouse anti-human CK (Dako) and Gnostics), which used a Z-Stack (5 images above and below goat anti-human CTSS (R&D Systems) overnight at 4° C. the focal plane, 0.3 um Steps, 20x objective) to generate a Sections were then washed in PBS and incubated with don maximal intensity projection (MIP) image of each acquired key-anti mouse HRP labeled secondary antibody (Jackson brain area. Tumor cells were detected via cell tracker green Immunoresearch, 1:200) in 0.25% PNB bufferin PBS for 1.5 (Invitrogen) and vasculature was visualized by Texas Red h followed by incubation with Alexa488 labeled tyramide Lectin (Vector Laboratories) injections. Tumor cells were (Invitrogen) at a 1:200 dilution in amplification buffer for 8 counted manually and their localization relative to the vascu min. Sections were then washed in PBS and incubated with lature was determined. donkey anti-goat Alexa568 (Molecular Probes) at a dilution 0170 For analysis of human samples, 5-10 fields of view of 1:500 in 0.25% PNB for 1 h at room temperature. Frozen were acquired using a 20x objective (total magnification sections that were used for Jam-B, Cldn3 and Ocln staining 200x) and a Zeiss Apotome to ensure cells were in the same were processed using a Ventana automated Staining device. optical section. The number of CK+ tumor cells and CD68+ The automated rehydration, citrate buffer-based antigen macrophages, and their relative CTSS intensities (CTSS retrieval, and blocking of unspecific protein binding and index) was evaluated using CellProfiler 2.0 software. A Cell endogenous peroxidase was followed by incubation with rat Profiler module was generated that allowed for the detection anti-mouse Jam-B (Pierce) primary antibody and goat anti of tumor cells and macrophages based on their DAPI and CK mouse Cd31 (R&D Systems), rabbitanti-mouse Cldn3 (Invit signal, or DAPI and CD68 signal, respectively. The CTSS rogen) and goat anti-mouse Cd31 (R&D Systems), or rabbit signal intensity was measured in the whole cell population anti-mouse Ocln (Invitrogen) and goat anti-mouse Cd31 (DAPI+) and associated with a specific cell type (macroph (R&D Systems) overnight at 4°C. Sections were thenwashed ages or tumor cells), and the proportion of CTSS signal asso in PBS and incubated with donkey anti-rat or donkey anti ciated with CK+ tumor cells or CD68+ macrophages was rabbit biotin labeled secondary antibody (Vector, 1:200) in calculated relative to the overall CTSS signal intensity in all PBS+0.03% Tween for 1.5 h followed by incubation with DAPI cells. Streptavidin-Cy5 (Invitrogen, 1:200) PBS+0.03% Tween for 20 min. Sections were then washed in PBS and incubated with donkey anti-goat Alexa568 (Molecular Probes) at a dilu Measurement of Vessel Permeability tion of 1:500 in 0.25% PNB for 1 hatroom temperature. After 0171 6-8 week old Athymic/nude mice were injected with washing in PBS, tissue sections were counterstained with Evans blue dye (30 mg/kg) into the tail vein. 30 mins after DAPI (5 mg/ml stock diluted 1:5,000 in PBS) for 5 min prior injection, mice were anesthetized and perfused with acidified to mounting with ProLong Gold Antifade mounting media fixative (1% PFA in 0.05 mM citrate buffer, pH3.5).30 mg of (Invitrogen). Apoptotic cells were stained via terminal dUTP brain tissue was incubated in 500 ul formamide (Sigma) to nick end labeling (TUNEL) following the manufacturers extract Evan's blue at 60° C. overnight. Absorbance was instructions (Trevigen), with the modification of using measured at 610 nm and 740 nm on a spectraMax 340pc plate Streptavidin-Cy5 (Invitrogen; 1:200) instead of Streptavidin FITC. reader (Molecular Devices). 0168 Tissue sections and cells on coverslips were visual In Vitro Blood-Brain Barrier Transmigration Assays ized under a Carl Zeiss Axioimager Z1 microscope equipped with an ApoTome.2 and a TissueCinostics stage to allow for 0172. In vitro blood-brain barrier (BBB) transmigration automated image acquisition. The analysis of proliferation assays were performed as previously described32. The arti and apoptosis were performed using Tissue(Quest analysis ficial BBB was formed with either HUVECs or HBMECs software (TissueCinostics) as previously described6, 55. All (20,000 cells/well) in co-culture with HA cells (100,000 parameters of metastatic outgrowth and angiogenesis were quantitated using MetaMorph software (Molecular Devices). cells/well) for 3 days on Transwell-inserts with 3 um fluorob Briefly, vasculature was visualized by Texas Red Lectin (Vec lock membranes. Cell-tracker green (CMFDA)-labeled Br-M tor Laboratories) injections or by staining of the endothelial Ctrlor Br-MCTSSKD cells (20,000 cells/well) were allowed cell marker CD34. Tumor cells were detected by their expres to transmigrate for 18 h through the artificial BBB towards a sion of the GFP reporter. The area covered by CD34 and GFP FBS gradient, in the presence or absence of VBY-999 (10 staining was quantified. To determine the number of tumor uM). Tumor cell transmigration through empty inserts cells that are present within an area of 1->4 average tumor cell (coated with gelatin and poly-L-lysine) or inserts coated with diameter, the blood vessel area was dilated by 1-4 average HUVECs, HBMECs or HAS alone were used to determine the tumor cell diameter with an increment of 1 tumor cell diam baseline migratory potential and the contribution of the single eter and the number of tumor cells in each area was deter cell types to BBB formation. Tumor cell transmigration was mined. Tumor cells that were localized outside an area of 4 stopped through fixation of the cells in 4% PFA. Cells were average tumor cell diameter were defined as >4 tumor cell counterstained with Hoechst dye (5 mg/ml stock diluted 1:5, diameter away from CD34+ blood vessels as illustrated in FIG. 4b. Vessel density was quantified as the area covered by 000 in PBS) for 5 min prior to mounting with ProLong Gold Texas Red Lectin relative to the area covered by DAPI. Antifade mounting media (Invitrogen). The number of trans 0169. To histologically quantify the percentage of intra migrated tumor cells was quantified by analyzing 200 fields vascular, extravasating or extravasated tumor cells (FIG. 5c). of views (FOVs) that were acquired with a 20x objective brain sections were stained for TUNEL+ cells to exclude (200x total magnification) using Tissue(Quest analysis Soft non-viable tumor cells from the analysis. Brain sections were ware (TissueCinostics). US 2016/0138113 A1 May 19, 2016 22
In Vitro and Cell-Based Cleavage Assays tion described herein. Accordingly, it is intended by the (0173 Recombinant inactive CTSS was obtained from appended claims to cover all such alternative aspects as fall R&D Systems. CTSS was activated at 50 ng/ul in 50 mM within the true spirit and scope of the invention. sodium acetate, 5 mM DTT, 0.25 MNaCl (pH 4.5) for 1.5 h 0178. In Table 1 below, gene symbol, gene name, P value at 37° C. For the in vitro cleavage reaction, activated CTSS and fold change of expression differences in the experimental was incubated with recombinant proteins in the presence or metastasis models are indicated in each column. Fold change absence of the cathepsin Sinhibitor VBY-999 (10 uM) for 0, is depicted Such that a negative (-) value is associated with 10 or 20 min in 50 mM sodium acetate, 5 mM DTT, 0.25 M downregulation in late-stage metastases, while a positive NaCl at pH 4.5 and pH 6.0. Details about the recombinant proteins used in the in vitro cleavage assay can be found in value is associated with upregulation in late-stage metastases. Table 6. The in vitro cleavage reaction was stopped by adding For Table 1a, positive values are associated with upregulation SDS sample buffer and reducing agent (Invitrogen) to each in early-stage metastases compared to normal lung, while reaction and the samples were boiled at 95°C. for 5 min. negative values are associated with downregulation in early Aliquots were subjected to western blot analysis as described stage lung metastases compared to normal lung. To identify above. Information about the antibodies can be found in Table Subtle changes in gene expression between normal lung and 5. All experiments were repeated independently at least three early stage lung metastases, a fold change cutoff of +1.5 was times. used in conjunction with a nominal P-value cut off of 0.05. 0.174 For cell-based cleavage assays, HBMECs were For every other analysis, a 2.0 fold change cutoff was used. P grown to 100% confluence in a 10 cm plate. Conditioned values were calculated as described in the methods using a media from Br-M cells was collected as described above.200 two-tailed Students t-test. ul of concentrated Br-MCM (collected from two 10 cm plates of confluent Br-M cells) was diluted in 6.5 ml PBS pH 6.0+ (0179. In Table 2 below, (a-c) Hazardratio,95% confidence 0.05 mM DTT for each 10 cm plate of HBMECs. The cleav interval (CI), and P values for genes identified from analysis age reaction was performed in the presence or absence of the of patient dataset GSE 12276, which are also listed in FIG.11. cathepsin Sinhibitor VBY-999 (10 uM) for 0h, 2 h, and 4 h. The “metastatic site association' column denotes whether a PBS pH 6.0+0.05 mM DTT was used as a control. The super gene shows association with patient MFS in multiple tissues, natant from the HBMEC cell layers was collected after the as summarized in FIG. 11f. Tumor-derived differentially indicated time points, concentrated and Subjected to western expressed genes (DEG) whose expression changes by stage in blot analysis as described above. the experimental model (FIG. 1c, Table 1) were assessed for Proliferation Assays MFS using a cox proportional hazards model, as described in the Online Methods. Hazard ratios with 95% CIS that do not 0175 Cell growth rate was determined using an MTT cell proliferation kit (Roche). Briefly, cells were plated in tripli cross 1.0 are considered significant. Nominal P values were cate in 96-well plates (2.5x10 for Br-MCtrland Br-MCTSS determined using Wald’s test. (d) Summary of the number of KD cells) in the presence or absence of 0.1-100 umVBY-999. DEGs associated with MFS at each organ site. These tests Reduction of the MTT substrate was detected by colorimetric aimed to address whether selecting genes for differential analysis using a plate reader as per the manufacturer's rec expression at the metastatic site enriched for genes associated ommended protocol. 10 ul of MTT labeling reagent was with site-specific MFS. These tests demonstrate that the set of added to each well and then incubated for 4 h at 37° C., genes differentially expressed in the bone is enriched for followed by the addition of 100 ul MTT solubilization reagent overnight. The mixture was gently resuspended and absor genes associated with bone MFS, while the brain and lung bance was measured at 595 nm and 750 nm on a spectraMax DEGs are not significantly enriched for genes associated with 340pc plate reader (Molecular Devices). brain or lung MFS. (e) Summary of differentially expressed genes in the bone, and their association with brain and lung Data Presentation and Statistical Analysis MFS. We sought to determine if the significant enrichment of 0176 Data are presented as means with standard error genes associated with bone MFS in the bone DEG set was (S.e.m.) or as statistical scatter plots using GraphPad Prism specific to the bone, or whether there was also an association Pro5. Numeric data were analyzed using unpaired two-tailed with brain and lung MFS. These hypergeometric tests dem Student's t-test unless otherwise noted. Kaplan-Meier sur onstrate that the set of genes differentially expressed in the vival curves, heatmaps and scatterplots were generated in R v bone is enriched for genes associated with bone MFS only, 2.15.2 using the base R graphics, gplots’ or 'ggplot2 pack and not genes associated with brain or lung MFS. Differences ages. P values were generated using the Log-Rank statistic for in the numbers between Table 1 and here are due to incom Kaplan-Meier Analysis and Wald’s test for the Hazard Ratio. plete overlap between coverage of genes on the HuMu ProtIn P<0.05 was considered as statistically significant. All code array, and genes in the GSE 12276 patient dataset as indicated. used to analyze the data and generate the plots is available at the following url: bitbucket.org/bowmanr/joycelab-humu P values were generated using a hypergeometric test. brain-met-ctss. 0180. In Table 3 below, for patients 1-6 matched pairs of 0177. While several aspects of the present invention have primary breast cancer and brain metastasis samples were been described and depicted herein, alternative aspects may available. For patients 7-13 only brain metastasis samples be effected by those skilled in the art to accomplish the same were available. MFS: metastasis-free survival. ER: estrogen objectives. Those skilled in the art will recognize, or be able receptor, PR: progesterone receptor, HER2: human epider to ascertain using no more than routine experimentation, mal growth factor receptor 2, Pos: positive, Neg: negative, many equivalents to the specific embodiments of the inven N/A: not assessed, MFS: metastasis-free survival.