Methods for treating minimal residual cancer

Abstract

To provide methods of treating minimal residual cancer in a subject.SOLUTION: The methods involve contacting disseminated cancer cells (DCCs) in a subject with a bone morphogenic protein 7 (BMP7) derivative protein, where the contacting induces or maintains dormancy in the contacted DCCs of the subject to treat minimal residual cancer in the subject. Also disclosed are methods that involve contacting DCCs in a subject with a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor selected from LY2, LY3, and LY4, where the contacting eradicates DCCs in the subject to treat minimal residual cancer in the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 648,166, filed March 26, 2018, which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support awarded under grants R01 CA109182, U54 CA16131, and P30 CA196521 from the National Institutes of Health / National Cancer Institute, and under grant BC 132674 from the Congressionally Directed Medical Research Programs of the U.S. Department of Defense. The U.S. Government has certain rights in this invention.

[0003] Field The present disclosure relates to methods of treating minimal residual cancer in a subject. [Background technology]

[0004] background Protein misfolding in the endoplasmic reticulum ("ER") lumen activates three major pathways, PERK, IRE1α, and ATF6, also known as the unfolded protein response ("UPR"), allowing cells to compensate for this stress and survive (Walter et al., "The Unfolded Protein Response: From Stress Pathway to Homeostatic Regulation," Science 334:1081-1086 (2011) and Ron et al., "Signal Integration In the Endoplasmic Reticulum Unfolded Protein Response," Nat. Rev. Mol. Cell Biol. 8:519-529 (2007)). Recent evidence suggests that in various types of cancer, the UPR, among other signals, is a mechanism that enables tumor cells to respond to oxidative conditions and the demands on the ER imposed by oncogenes and the increased transcriptional load caused by hypoxia (Blais et al., "Activating Transcription Factor 4 is Translationally Regulated by Hypoxic Stress," Mol. Cell. Biol. 24:7469-7482 (2004); Chevet et al., "Endoplasmic Reticulum Stress-Activated Cell Reprogramming in Oncogenesis," Cancer Discov. 5:586-597 (2015); Tameire et al., "Cell Intrinsic and Extrinsic Activators of the Unfolded Protein Response in Cancer: Mechanisms and Targets for Therapy," Semin. Cancer Biol. 33:3-15 (2015); Hart et al., “ER Stress-Mediated Autophagy Promotes Myc-Dependent Transformation and Tumor Growth,” J. Clin. Invest. 122:4621-4634 (2012); Martin-Perez et al., “Activated ERBB2 / HER2 Licenses Sensitivity to Apoptosis Upon Endoplasmic Reticulum Stress Through a PERK-Dependent Pathway,” Cancer Res. 74:1766-1777 (2014); Rajasekhar et al., “Postgenomic Global Analysis of Translational Control Induced by Oncogenic Signaling,” Oncogene 23:3248-3264 (2004); Rajasekhar et al., “Oncogenic Ras and Akt Signaling Contribute to Glioblastoma Formation by Differential Recruitment of Existing mRNAs to Polysomes,” Mol. Cell 12:889-901 (2003); Rojo et al., “4E-Binding Protein 1, A Cell Signaling Hallmark in Breast Cancer that Correlates With Pathologic Grade and Prognosis,” Clin. Cancer Res. 13:81-89 (2007);およびSequeira et al., “Inhibition of eIF2alpha Dephosphorylation Inhibits ErbB2-Induced Deregulation of Mammary Acinar Morphogenesis,” BMC Cell Biol.10:64 (2009)). Pathways activated by oncogenes increase ER client protein load by activating mTOR signaling and initiating translation (Hart et al., “ER Stress-Mediated Autophagy Promotes Myc-Dependent Transformation and Tumor Growth,” J. Clin. Invest. 122:4621-4634 (2012); Ozcan et al., “Loss of the Tuberous Sclerosis Complex Tumor Suppressors Triggers the Unfolded Protein Response to Regulate Insulin Signaling and Apoptosis,” Mol. Cell 29:541-551 (2008); and Tameire et al., “Cell Intrinsic and Extrinsic Activators of the Unfolded Protein Response in Cancer: Mechanisms and Targets for Therapy,” Semin. Cancer Biol. 33:3-15 (2015)). The PERK and IRE1α-XBP-1 pathways have further been shown to contribute to adaptation to hypoxia and microenvironmental stress (Bi et al., "ER Stress-Regulated Translation Increases Tolerance to Extreme Hypoxia and Promotes Tumor Growth," EMBO J. 24:3470-3481 (2005); Blais et al., "Activating Transcription Factor 4 is Translationally Regulated by Hypoxic Stress," Mol. Cell. Biol. 24:7469-7482 (2004); Chen et al., “XBP1 Promotes Triple-Negative Breast Cancer by Controlling the HIF1alpha Pathway,” Nature 508:103-107 (2014); Romero-Ramirez et al., “X box-Binding Protein 1 Regulates Angiogenesis in Human Pancreatic Adenocarcinomas,” Transl. Oncol. 2:31-38 (2009); Rouschop et al. al., “The Unfolded Protein Response Protects Human Tumor Cells During Hypoxia Through Regulation of the Autophagy Genes MAP1LC3B and ATG5,” J. Clin. Invest. 120:127-141 (2010); Schewe et al., “ATF6alpha-Rheb-mTOR Signaling Promotes Survival of Dormant Tumor Cells In Vivo,” Proc. Nat'l. Acad. Sci. USA 105:10519-10524 (2008); and Ye et al., "The GCN2-ATF4 Pathway is Critical for Tumor Cell Survival and Proliferation in Response to Nutrient Deprivation," EMBO J. 29:2082-2096 (2010)), suggesting that the UPR may enable adaptation to a changing environment.

[0005] PERK activation orchestrates antioxidant and autophagy responses to protect mammary epithelial cells despite loss of attachment to the basement membrane (Avivar-Valderas et al., “PERK Integrates Autophagy and Oxidative Stress Responses to Promote Survival During Extracellular Matrix Detachment,” Mol. Cell. Biol. 31:3616-3629 (2011)). This survival response involves the ATF4 and CHOP transcriptional programs (Avivar-Valderas et al., "PERK Integrates Autophagy and Oxidative Stress Responses to Promote Survival During Extracellular Matrix Detachment," Mol. Cell. Biol. 31:3616-3629 (2011)) and is coupled to rapid activation of the LKB1-AMPK-TSC2 pathway, which inhibits mTOR (Avivar-Valderas et al., "Regulation of Autophagy during ECM Detachment is Linked to a Selective Inhibition of mTORC1 by PERK," Oncogene 32(41):4932-40 (2013)).Human DCIS lesions show enhanced PERK phosphorylation and autophagy (Avivar-Valderas et al., "PERK Integrates Autophagy and Oxidative Stress Responses to Promote Survival During Extracellular Matrix Detachment," Mol. Cell. Biol. 31:3616-3629 (2011) and Espina et al., "Malignant Precursor Cells Pre-Exist in Human Breast DCIS and Require Autophagy for Survival," PloS One 5:e10240 (2010)), and conditional ablation of PERK in breast epithelium delayed the development of HER2-driven breast cancer (Bobrovnikova-Marjon et al., "PERK Promotes Cancer Cell Proliferation and Tumor Growth by Limiting Oxidative DNA Damage," Oncogene 29:3881-3895). (2004) and Bobrovnikova-Marjon et al., "PERK-Dependent Regulation of Lipogenesis During Mouse Mammary Gland Development and Adipocyte Differentiation," Proc. Nat'l. Acad. Sci. USA 105:16314-16319 (2008)). Furthermore, HER2 increases proteotoxicity levels in tumor cells, activating JNK and IRE signaling and HER2. +This allows cancer cells to respond to this stress (Singh et al., "HER2-mTOR Signaling-Driven Breast Cancer Cells Require ER-Associated Degradation to Survive," Sci. Signal. 8:ra52 (2015)). Accordingly, the cBIO database (Cerami et al., "The cBio Cancer Genomics Portal: An Open Platform for Exploring Multidimensional Cancer Genomics Data," Cancer Discov. 2:401-404 (2012)) shows that approximately 14% of HER2-amplified human breast tumors exhibit upregulation of PERK mRNA, suggesting that HER2 + Further supporting the concept that tumors may depend on PERK and / or other UPR pathways for survival.

[0006] Dormant (quiescent) tumor cells are also known to depend on PERK and ATF6 signaling for survival (Ranganathan et al., “Dual Function of Pancreatic Endoplasmic Reticulum Kinase in Tumor Cell Growth Arrest and Survival,” Cancer Res. 68:3260-3268 (2008); Ranganathan et al., “Functional Coupling of p38-Induced Up-Regulation of BiP and Activation of RNA-Dependent Protein Kinase-Like Endoplasmic Reticulum Kinase to Drug Resistance of Dormant Carcinoma Cells,” Cancer Res. 66:1702-1711 (2006); and Schewe et al., “ATF6alpha-Rheb-mTOR Signaling Promotes Survival of Dormant Tumor Cells In Vivo,” Proc. Nat'l. Acad. Sci. USA 105:10519-10524 (2008)). Quiescent pancreatic disseminated cancer cells ("DCCs") in the liver also exhibited a PERK-dependent UPR associated with loss of E-cadherin expression and downregulation of MHC-I, favoring immune evasion during dormancy (Pommier et al., "Unresolved Endoplasmic Reticulum Stress Engenders Immune-Resistant, Latent Pancreatic Cancer Metastases," Science 360(6394):eaao4908 (2018), incorporated herein by reference in its entirety). In the MMTV-HER2 model, quiescent DCCs in the bone marrow and lung were also found to be E-cadherin negative (Harper et al., "Mechanism of Early Dissemination and Metastasis in Her2 +The association with UPR was not examined. Collectively, these data suggest that UPR may act as a survival mechanism for DCCs' adaptability to stress and the immune microenvironment.

[0007] The present disclosure is directed to overcoming deficiencies in the art. Summary of the Invention

[0008] overview One aspect of the present disclosure relates to a method for treating minimal residual cancer in a subject, the method comprising contacting disseminated cancer cells (DCCs) in the subject with a bone morphogenetic protein 7 ("BMP7") derivative protein, thereby inducing or maintaining dormancy of the contacted DCCs in the subject and treating the minimal residual cancer in the subject. The method of this aspect can be used to prevent minimal residual cancer from progressing to invasive growth in the subject.

[0009] Another aspect relates to a method of treating minimal residual cancer in a subject, the method comprising contacting disseminated cancer cells (DCCs) in the subject with a protein kinase RNA-like endoplasmic reticulum kinase ("PERK") inhibitor selected from LY2, LY3, and LY4, thereby eradicating the DCCs in the subject and treating the minimal residual cancer in the subject.

[0010] Yet another aspect relates to a method of treating late-stage cancer in a subject, the method comprising contacting disseminated cancer cells (DCCs) in the subject with a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor selected from LY2, LY3, and LY4, thereby eradicating the DCCs in the subject and treating the late-stage cancer in the subject.

[0011] In particular, we demonstrate that LY4, a selective and potent PERK inhibitor, can disrupt HER2-induced metastasis as a result of its ability to specifically eradicate dormant DCCs. In this disclosure, PERK inhibitors represent a novel strategy that targets single dormant cells at the minimal residual disease stage, helping to prevent lethal metastasis both as a single agent and in combination with antiproliferative therapy. We also demonstrate that osteogenic inducer protein can induce dormancy in disseminated tumor cells. [The present invention 1001] contacting disseminated cancer cells (DCCs) in a subject with a bone morphogenetic protein 7 ("BMP7") derivative protein, thereby inducing or maintaining dormancy of the contacted DCCs in the subject to treat minimal residual cancer in the subject; 1. A method of treating minimal residual cancer in a subject, comprising: [The present invention 1002] 1001. The method of claim 1001, wherein said subject has been diagnosed with breast cancer, multiple myeloma, lung cancer, non-small cell lung cancer, brain cancer, cervical cancer, mantle cell lymphoma, leukemia, hepatocellular carcinoma, prostate cancer, melanoma, skin cancer, head and neck cancer, thyroid cancer, glioblastoma, neuroblastoma, or colorectal cancer. [The present invention 1003] 1002. The method of claim 1002, wherein said cancer is a breast cancer selected from invasive breast cancer, ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and inflammatory breast cancer. [The present invention 1004] The breast cancer is HER2 + The method of the present invention 1003, wherein the cancer is breast cancer. [The present invention 1005] The method of any of claims 1001 to 1004, wherein said subject has been diagnosed with disseminated tumor cells and / or non-metastatic cancer. [The present invention 1006] The method of any one of claims 1001 to 1005, wherein the BMP7 derivative is BMP7-F9. [The present invention 1007] administering to said subject a chemotherapeutic agent, an immunotherapeutic agent, an epigenetic agent, or ionizing radiation. The method of any one of 1001 to 1006 of the present invention, further comprising: [The present invention 1008] 1007. The method of claim 1007, wherein said subject is administered a chemotherapeutic agent, and said chemotherapeutic agent is an anti-HER2 chemotherapeutic agent selected from trastuzumab (Herceptin®) and lapatinib (Tykerb®). [The present invention 1009] 1007. The method of claim 1007, wherein said subject is administered a chemotherapeutic agent, and said chemotherapeutic agent is selected from an anthracycline, a taxane, a kinase inhibitor, an antibody, a fluoropyrimidine, and a platinum drug. [The present invention 1010] 1007. The method of claim 1007, wherein said subject is administered an immunotherapeutic agent, and said immunotherapeutic agent is selected from an immune checkpoint inhibitor, an interferon, or a tumor vaccine. [The present invention 1011] The method of claim 1007, wherein an epigenetic agent is administered to the subject, and the epigenetic agent is selected from a histone deacetylase (HDAC) inhibitor, 5-azacytidine, retinoic acid, arsenic trioxide, a Zeste2 enhancer polycomb repressive complex 2 subunit (EZH2) inhibitor, a bromodomain (BRD) inhibitor, and derivatives thereof. [The present invention 1012] The method of any of claims 1001 to 1011, wherein the contacting step is carried out by administering the BMP7 derivative protein to the subject. [The present invention 1013] detecting the presence of DCC in said subject prior to said contacting step. Any of the methods of 1001 to 1012 of the present invention, further comprising: [The present invention 1014] The DCC is NR2F1 + The method of the present invention 1013. [The present invention 1015] The DCCs were bone morphogenetic protein receptor-positive (BMPR + "). The method of the present invention 1013 or 1014. [The present invention 1016] 1016. The method of any one of claims 1013 to 1015, wherein the DCC is phospho-PERK active. [The present invention 1017] contacting DCCs in said subject with a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor, a MEK inhibitor, a CDK4 / 6 inhibitor, or any combination thereof. Any of the methods of 1001 to 1016 of the present invention, further comprising: [The present invention 1018] The method of claim 1017, wherein said contacting step is carried out by administering a PERK inhibitor to said subject. [The present invention 1019] The method of claim 1017 or claim 1018, wherein said contacting step is carried out with a PERK inhibitor selected from LY2, LY3, LY4, and combinations thereof. [The present invention 1020] Any of the methods of claims 1017 to 1019, wherein the contacting step is carried out using a PERK inhibitor that does not inhibit EIF2AK1, EIF2AK2, or EIF2AK4. [The present invention 1021] 1017. The method of claim 1017, wherein said contacting step is carried out by administering a MEK inhibitor to said subject. [The present invention 1022] The method of the present invention 1021, wherein the MEK inhibitor is selected from PD184352, PD318088, PD98059, PD334581, RDEA119 / BAY 869766. [The present invention 1023] 1017. The method of claim 1017, wherein said contacting step is carried out by administering a CDK4 / 6 inhibitor to said subject. [The present invention 1024] 1023. The method of claim 1023, wherein said CDK4 / 6 inhibitor is selected from abemaciclib (LY2835219), palbociclib (PD0332991), and ribociclib (LEE011). [The present invention 1025] The method of any one of claims 1001 to 1024, wherein the subject is a human. [The present invention 1026] selecting a subject in cancer remission prior to said contacting step. Any of the methods of 1001 to 1025 of the present invention further comprising: [The present invention 1027] contacting disseminated cancer cells (DCCs) in a subject with a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor selected from LY2, LY3, and LY4, thereby eradicating DCCs in the subject and treating minimal residual cancer in the subject; 1. A method of treating minimal residual cancer in a subject, comprising: [The present invention 1028] 1027. The method of claim 1027, wherein said subject has been diagnosed with breast cancer, multiple myeloma, lung cancer, non-small cell lung cancer, brain cancer, cervical cancer, mantle cell lymphoma, leukemia, hepatocellular carcinoma, prostate cancer, melanoma, skin cancer, head and neck cancer, thyroid cancer, glioblastoma, neuroblastoma, or colorectal cancer. [The present invention 1029] 1028. The method of claim 1028, wherein said cancer is a breast cancer selected from invasive breast cancer, ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and inflammatory breast cancer. [The present invention 1030] The breast cancer is HER2 + The method of claim 1029, wherein the cancer is breast cancer. [The present invention 1031] The method of claim 1027, wherein said subject has been diagnosed with disseminated tumor cells and / or non-metastatic cancer. [The present invention 1032] administering to said subject a chemotherapeutic agent, an immunotherapeutic agent, an epigenetic agent, or ionizing radiation. Any of the methods of inventions 1027 to 1031, further comprising: [The present invention 1033] The method of claim 1032, wherein said subject is administered a chemotherapeutic agent, and said chemotherapeutic agent is an anti-HER2 chemotherapeutic agent selected from trastuzumab (Herceptin®) and lapatinib (Tykerb®). [The present invention 1034] 1033. The method of claim 1032, wherein said subject is administered a chemotherapeutic agent, and said chemotherapeutic agent is selected from an anthracycline, a taxane, a kinase inhibitor, an antibody, a fluoropyrimidine, and a platinum drug. [This invention 1035] 1033. The method of claim 1032, wherein said subject is administered an immunotherapeutic agent, and said immunotherapeutic agent is selected from an immune checkpoint inhibitor, interferon, or a tumor vaccine. [The present invention 1036] The method of claim 1032, wherein an epigenetic agent is administered to the subject, and the epigenetic agent is selected from a histone deacetylase (HDAC) inhibitor, 5-azacytidine, retinoic acid, arsenic trioxide, a Zeste2 enhancer polycomb repressive complex 2 subunit (EZH2) inhibitor, a bromodomain ("BRD") inhibitor, and derivatives thereof. [This invention 1037] Any of the methods of claims 1027 to 1036, wherein the contacting step is carried out by administering the PERK inhibitor to the subject. [The present invention 1038] detecting the presence of DCC in said subject prior to said contacting step. Any of the methods of claims 1027 to 1037, further comprising: [This invention 1039] The DCC is NR2F1 + The method of the present invention 1038. [The present invention 1040] The method of claim 1038 or claim 1039, wherein said DCC is phospho-PERK active. [The present invention 1041] The DCCs were bone morphogenetic protein receptor-positive (BMPR + Any of the methods of present inventions 1038 to 1040, wherein [The present invention 1042] contacting DCCs in said subject with a bone morphogenetic protein 7 (BMP7) derivative protein. The method of the present invention 1041 further comprising: [This invention 1043] The method of claim 1042, wherein the step of contacting DCCs in the subject with a BMP7 derivative protein is carried out by administering the BMP7 derivative protein to the subject. [This invention 1044] The method of claim 1042 or claim 1043, wherein the BMP7 derivative protein is BMP7-F9. [This invention 1045] The method of any one of claims 1027 to 1044, wherein the PERK inhibitor does not inhibit EIF2AK1, EIF2AK2, or EIF2AK4. [The present invention 1046] The method of any one of claims 1027 to 1045, wherein the subject is a human. [This invention 1047] selecting a subject in cancer remission prior to said contacting step. Any of the methods of claims 1027 to 1046, further comprising: [Brief explanation of the drawings]

[0012] [Figure 1A] Figures 1A-1C show that quiescent disseminated HER2+ cells exhibit high levels of ER stress pathway activation. Figure 1A shows images of lung sections from MMTV-HER2 animals stained for HER2, Ki67 (proliferation), and GADD34 (ER stress). The graph in Figure 1A shows quantification of cells / metastases positive for both markers as a percentage of total cells. Figure 1B shows images of human breast cancer metastases from different locations (lymph nodes, liver, and lungs) stained for cytokeratin, Ki67 (proliferation), and GADD34 (ER stress). The graph in Figure 1B shows quantification of cells / metastases positive for both markers as a percentage of total cells. Figure 1C shows hierarchical clustering of high-throughput target gene expression (vertical) profiles of single cells (lung disseminated tumor cells ("DTCs")) (horizontal). [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2A]Figures 2A-2G demonstrate that PERK inhibition is upregulated in patients with HER2+ cancer cells. Figure 2A is a flow diagram of the steps performed in single-cell gene expression analysis using Fluidigm C1 and Biomark HD. A total of 255 DCC and 90 primary tumor ("PT") cells were analyzed. Figure 2B shows a list of genes analyzed by high-throughput qPCR. Figure 2C is an immunoblot showing inhibition of PERK phosphorylation by LY series inhibitors (LY2, LY3, and LY4) and GSK2656157 (2 μM) in MCF10A-HER2 cells stressed in suspension for 24 hours. * indicates a nonspecific band. Figure 2D shows LY4 dose-response cell viability curves (Cell Titer Blue, CTB) of MCF10A-HER2 cells after 48 hours in the absence (-) or presence of stress (low-dose thapsigargin, Tg 2 nM). The dotted line indicates the IC50 (≈9 nM). Figure 2E shows the kinase selectivity of the PERK inhibitors LY4, LY2, LY3, and GSK2656157, as assessed by enzyme biochemical assay. Figure 2F shows the effect of LY4 on total bone marrow cells (in two hind limbs) of MMTV-HER2 females treated for two weeks. Figure 2G shows the effect of LY4 on total leukocytes of MMTV-HER2 females treated for two weeks. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E-1] See legend to Figure 2A. [Figure 2E-2] See legend to Figure 2A. [Figure 2E-3] See legend to Figure 2A. [Figure 2E-4] See legend to Figure 2A. [Figure 2F] See legend to Figure 2A. [Figure 2G] See legend to Figure 2A. [Figure 3A]Figures 3A-3G show that PERK inhibition by LY4 reduces metastatic disease in the lung and bone marrow at the single disseminated tumor cell level. Figure 3A is an immunoblot showing inhibition of PERK phosphorylation (T980) by the PERK inhibitor LY4 (2 μM) in MCF10A-HER2 cells serum-starved overnight and then treated with EGF (100 ng / ml) for 15 minutes. Figure 3B shows immunohistochemistry ("IHC") of pancreatic and mammary gland sections using antibodies against p-PERK and p-EIF2α. The inset shows a higher magnification. The scale bar is 100 μm. Figure 3C shows images and quantification (right graph) of macrometastases (>100 cells) detected by H&E staining and quantified in five lung sections / animal (n=16). Scale bar: 100 μm. p by Mann-Whitney test. Figure 3D shows images and quantification (right graph) of micrometastases (2-100 cells) detected by IHC staining with an anti-HER2 antibody and quantified (±sd) in each lung section / animal (n=6). Scale bar: 25 μm. p by Mann-Whitney test. Figure 3E shows images and quantification (right graph) of solitary disseminated tumor cells (DTCs) detected by IHC staining for HER2, classified as P-Rb+ or P-Rb-, and quantified (±sd) in each lung section (n=6). Scale bar: 25 μm. p by Mann-Whitney test. Arrows and circles in the images indicate solitary DTCs. Figure 3F shows images and quantification (right graph) of disseminated tumor cells in bone marrow, detected by IF staining for CK8 / 18 and HER2 on cytospins from mature hematopoietic cell-depleted bone marrow tissue (n=8). Scale bar: 25 μm. p is based on the Mann-Whitney test. Arrows indicate Her2+ cells. Figure 3G shows representative images of ZR75.1 HER2+ cells engineered to express Dendra-tagged H2B protein and seeded at low density (single cells) on Matrigel. On day 0, the Dendra tag (green fluorescence) was photoconverted to red fluorescence by a single UV light pulse and used as a measure of quiescence. Wells were treated with vehicle (DMSO) or LY4 (2 μM) from days 2 to 8.The graph shows the percentage of viable cells (±sd) measured on day 8 (n=4). p is based on Student's t-test. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 3F] See legend to Figure 3A. [Figure 3G] See legend to Figure 3A. [Figure 4A] Figures 4A-4F show the effect of LY4 treatment on metastasis and circulating tumor cells ("CTCs"). Figure 4A shows the relative area of single macrometastases in vehicle- and LY4-treated animals (n = 21 and 15, respectively). p is by Mann-Whitney test. Figure 4B is a graph showing quantification of circulating tumor cells / ml of blood by HER2 staining of cytospins. Figure 4C shows images and a graph showing the percentage of P-Rb+ micrometastases in each lung section / animal (n = 4 and 6, respectively). Figure 4D is an image showing 100% photoconverted ZR75.1-H2B-Dendra from green fluorescence to red fluorescence on day 0 after seeding in 3D Matrigel. In Figure 4E, ZR75.1-H2B-Dendra photoconverted cells were seeded at low (single cell) density or high density. The graph shows the percentage (±sd) of red label retention for cells seeded as single cells or at high density (n=4). p by Student's t-test. Figure 4F is the same as Figure 4D, but cells seeded at high density were treated with vehicle (DMSO) or LY4 (2 μM) from day 2 to day 8. The graph shows the percentage (±sd) of viable colonies measured on day 8 (n=4). p by Student's t-test. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E]See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 5A] Figures 5A-5C demonstrate that PERK inhibition is upregulated in Her2+ cells. Figure 5A shows that PERK (EIF2AK3) is upregulated in a subpopulation of HER2+ breast cancer patients. Analysis of TCGA breast cancer data (58 tumors) from HER2+ cases using cBioPortal. Figure 5B shows representative images of carmine staining of whole-mount FVB normal mammary glands compared to vehicle- and LY4-treated MMTV-neu mammary gland whole-mounts. Figure 5C shows quantification of the histological structures present in H&E-stained mammary gland sections (top images range from normal empty ducts to DCIS-like mammary intraepithelial neoplasia; bottom images are at higher magnification). [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6A] Figures 6A-6C demonstrate that the PERK inhibitor LY4 results in mammary gland "normalization" in the MMTV-HER2+ breast cancer model. Figure 6A shows representative images of carmine-stained and H&E-stained mammary gland sections from whole-mount mammary glands derived from vehicle- and LY4-treated animals. Scale bars are 100 µm. Figure 6B shows quantification (±s.e.m.) of the histological structures (empty ducts, obstructed ducts, obstructed hyperplasia, and DCIS-like mammary intraepithelial neoplasia) present in H&E-stained mammary gland sections (n = 50 / animal, n = 13 animals) from vehicle- and LY4-treated animals. Statistical significance (p) was calculated by the Mann-Whitney test. Figure 6C shows IHC of the epithelial luminal marker, cytokeratin 8 / 18 (CK8 / 18), and the myoepithelial marker, smooth muscle actin ("SMA"), in the mammary gland sections. The graph shows the scores for CK8 / 18+ and SMA+ structures for each animal (n=12). p is based on the Mann-Whitney test. Scale bar is 75 μm. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7A]Figures 7A–7F show the effects of LY4 treatment on P-PERK levels, P-histone H3 levels, and tumor size. Figure 7A is a Western blot for P-PERK levels in MMTV-neu tumor lysates from vehicle- and LY4-treated animals. Figure 7B shows tumor volumes (mm3) in vehicle-treated females (top) and LY4-treated females (bottom). Each line represents a tumor. Figure 7C shows the percentage tumor size reduction in LY4-treated females that showed tumor regression. Each line represents a tumor and an animal. Figure 7D shows representative images and quantification (right graph) of IHC for P-histone H3 in mammary tumor sections. p is calculated by the Mann-Whitney test. In Figure 7E, HER2-overexpressing ZR75.1 cells were seeded in Matrigel, and after the establishment of acini (day 10), wells were treated with vehicle (control) or LY4 (2 μM) for 10 days. The graph shows the percentage (±sd) of cleaved caspase-3-positive cells in each acinus (n=20). p is by Student's t-test. In Figure 7F, MCF10A-HER2 cells were seeded on Matrigel, and after acinar formation (day 4), wells were treated with vehicle (control) or LY4 (2 μM) for 10 days. The graph shows the percentage (±sd) of P-histone H3-positive cells in each acinus (n=20). p is by Student's t-test. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 7F] See legend to Figure 7A. [Figure 8A]Figures 8A–8D show that PERK inhibition prevents tumor growth in MMTV-HER2+ females. In Figure 8A, MMTV-neu females (24–32 weeks old) with overt tumors were injected daily for 2 weeks with vehicle or LY4 (50 mpk). The graph shows the percentage change in tumor size (±sd) in vehicle- and LY4-treated animals (n=16). *P<0.05, p<0.05). Figure 8B shows the final tumor volume (mm3). The whiskers represent the minimum and maximum values of the data (n=16). *P<0.05). Figure 8C shows representative IHC results for TUNEL staining to measure apoptosis levels within tumor sections. Scale bars are 10 μm and 50 μm. The graph shows the percentage of TUNEL-positive cells in vehicle- and LY4-treated tumor sections (n=5). *P<0.05). In Figure 8D, HER2+ MCF10A-HER2 or SKBR3 cells were seeded on Matrigel, and after acini formation (day 4), wells were treated with vehicle (control) or LY4 (2 μM) for 10 days. The graph shows the percentage (±sd) of cleaved caspase-3-positive cells in each acinus (n=20). p is by Student's t-test. Representative confocal images of MCF10A-HER2 acini stained for cleaved caspase-3. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 9A]Figures 9A-9F show that LY4 treatment reduces the levels of phospho-HER2 and downstream signaling pathways. Figure 9A shows representative images of IHC for P-HER2, P-PERK, and P-EIF2α in MMTV-HER2 mammary tumor sections. Note that the P-HER2-positive rim overlaps with P-PERK and P-EIF2α staining. Scale bar: 100 μm. Figure 9B shows hierarchical clustering of high-throughput target gene expression (vertical) profiles of single cells (primary mammary tumors) (horizontal) from MMTV-HER2 females. Figure 9C shows representative IHC staining for P-HER2 and total HER2 in vehicle- and LY4-treated mammary tumors. Graphs show P-HER2 scores for vehicle- and LY-treated tumors. Quantification of P-HER2 levels in tumor sections was by IHC intensity and area scoring (n=11) (see Figure 10A). Scale bar: 50 μm. p is based on the Mann-Whitney test. In Figure 9D, MCF10A-HER2 cells were starved overnight and treated with + / - LY4 (2 μM), followed by the addition of + / - EGF (100 ng / ml) for 15 minutes before harvesting. Levels of P-HER2, P-EGFR, P-AKT, P-S6, and P-ERK, as well as total HER2 and EGFR, were assessed by Western blot. GAPDH and β-TUB were used as loading controls. Three representative blots are shown. Densitometry analysis of P-HER2 (n = 3) (±s.d.). p is based on Student's t-test. In Figure 9E, MCF10A-HER2 cells were treated as in Figure 9D, and a surface receptor biotinylation assay was performed. Surface levels of total HER2 and P-HER2 were assessed. Densitometry of P-HER2 is shown. In Figure 9F, MCF10A-HER2 cells were treated as in Figure 9D and a reversible surface receptor biotinylation assay was performed. The endocytosis levels of total HER2 and P-HER2 were assessed. One of two experiments is shown. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 9E]See legend to Figure 9A. [Figure 9F] See legend to Figure 9A. [Figure 10A] Figures 10A-10C show quantification of P-HER2 levels in MCF10A-HER2 cells. Figure 10A shows the scoring system used to quantify P-HER2 levels in breast tumor sections. The IHC P-HER2-positive area was multiplied by its intensity score according to the established score shown in these representative images. Scale bar: 100 μm. In Figure 10B, MCF10A-HER2 cells were starved overnight and treated with + / - LY4 (2 μM), followed by the addition of + / - EGF (100 ng / ml) for 20 minutes before harvesting. P-HER2 / Y1112 and P-HER2 / Y877 levels were assessed by Western blot. GAPDH and HSP90 were used as loading controls. Figure 10C shows the extract input used for the surface biotinylation assay. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 11A]Figures 11A–11E show that sequential CDK4 / 6 inhibition followed by PERK inhibition enhances the anti-metastatic effect of LY4. Figure 11A is a schematic diagram of an in vivo experiment designed to evaluate sequential treatment with abemaciclib and LY4 in the MMTV-neu / HER2+ mouse model. MMTV-neu / HER2+ female mice (24 weeks old) were treated daily for 4 weeks with the CDK4 / 6 inhibitor abemaciclib (50 mpk), followed by treatment with LY4 (50 mpk). Figure 11B shows a series of fluorescent IHC images of HER2, Ki67 (proliferation), and GADD34 (ER stress) in tumor sections. Scale bar: 100 μm. Arrows indicate hyperfluorescence. Figure 11C shows the number of macrometastases (>100 cells) detected by H&E staining and quantified in five lung sections per animal (n=8). p is based on the Mann-Whitney test. Figure 11D is a graph showing the number of micrometastases (2–100 cells) detected by IHC staining with an anti-HER2 antibody and quantified in each lung section / animal (±sd) (n=8). p is based on the Mann-Whitney test. Figure 11E is a graph showing the number of single disseminated tumor cells detected by IHC staining for HER2, classified as Ki67+ or Ki67-, and quantified in each lung section (±sd) (n=8). p is based on the Mann-Whitney test. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 11D] See legend to Figure 11A. [Figure 11E] See legend to Figure 11A. [Figure 12A]Figures 12A-12G present proposed monotherapy or combination therapies involving the use of LY4, as well as experiments demonstrating that treatment of melanoma cells with the CDK4 / 6 inhibitor abemaciclib in combination with LY4 differentially affects cell viability in vitro in 2D and 3D cultures. Figure 12A is a schematic diagram of the basic principle of abemaciclib and LY4 combination. Figure 12B is a bar graph showing the results of in vitro treatment of Braf-mutant melanoma cells (WM35) with 0 nM, 10 nM, or 50 nM abemaciclib for 1 week, followed by 2 μM LY4 for 48 hours. Figure 12C includes images of DAPI-stained cells after 1 week of abemaciclib pretreatment followed by 2 μM LY4 treatment. 5,000 cells were seeded onto Matrigel. In Figures 12D-12E, WM35 melanoma cells were pretreated with abemaciclib for 5 weeks and then treated with LY4 in complete medium and abemaciclib. Cells were stained with trypan blue to identify viable cells. Figure 12D shows graphs of abemaciclib-sensitive cells. Figure 12E shows graphs of abemaciclib-resistant cells. Figure 12F shows DAPI-stained images of cells pretreated with abemaciclib for 5 weeks, then co-treated with 2 μM LY4 and abemaciclib. 1000 cells were seeded onto Matrigel. Figure 12G suggests that upon induction of growth arrest with abemaciclib, the cells upregulate a PERK target (GADD34), which may explain why the cells are sensitive to LY4. Abemaciclib-naïve or -resistant (R) WM35 melanoma cells were treated with vehicle (-) or 150 nM and 300 nM abemaciclib for 24 hours in culture. Cells were then lysed, and GADD34 expression was examined by Western blot. Tubulin expression was used as a loading control. Note that GADD34 was upregulated in abemaciclib-naïve cells, suggesting PERK activation. Resistant cells appeared to exhibit higher GADD34 levels that did not change or decrease after additional abemaciclib treatment. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 12E] See legend to Figure 12A. [Figure 12F-1] See legend to Figure 12A. [Figure 12F-2] See legend to Figure 12A. [Figure 12G] See legend to Figure 12A. [Figure 13A] Figures 13A-13C demonstrate the effects of BMP7-F9 on the ERK / p38 activity ratio and various mRNAs associated with dormancy signature genes. Figure 13A shows that treatment with 2 ng / ml, 5 ng / ml, and 10 ng / ml BMP7-F9 (second, third, and fourth gray bars, respectively; control, first black bar) reduced the ERK / p38 activity ratio compared to the control, as determined by Western blot in HEp3 HNSCC cells. The effect on the ERK / p38 activity ratio is observed after 2, 6, and 24 hours (second to fourth vertical groups). ERK activity is stimulated by BMP7 for the first 30 minutes (first vertical set). Figure 13B shows that BMP7-F9 treatment (10 ng / ml BMP7-F9 for 24 hours) induces DEC2, p53, and p27 mRNAs, which encode dormancy signature genes. Figure 13C shows that BMP7-F9 treatment of the same cells induces nuclear accumulation of the potent dormancy-inducing transcription factor NR2F1, as determined by immunofluorescence (10 ng / ml for 24 hours). Arrows indicate NR2F1 fluorescence. Differences between Figures 13A and 13B are p<0.05, calculated by Student's t-test. These data support the hypothesis that BMP7-F9 is a potent inducer of dormancy genes that are upregulated in spontaneous dormant DCCs or induced by reprogramming or TGFβ2 signaling in the bone marrow. [Figure 13B] See legend to Figure 13A. [Figure 13C] See legend to Figure 13A. [Figure 14A]Figures 14A-14E show how BMP7-F9 induces growth arrest in T-HEp3 cells in vitro and in vivo. Figure 14A shows that BMP7-F9 treatment of T-HEp3 cells inhibits proliferation for 48 hours in vitro, as determined by cell titer blue assay (RFU, relative fluorescence units). Figure 14B is a schematic diagram of the in vivo experimental procedure used in Figures 14C-14D. T-HEp3 cells were pretreated with BMP7-F9 for 24 hours in vitro and then inoculated into the chick embryo chorioallantoic membrane ("CAM") (Figure 14C). After daily treatment with vehicle or BMP7-F9 (50 ng / ml) in vivo, tumors were harvested, and the number of HEp3 HNSCC cells was quantified (Figure 14D) and the level of P-H3 was quantified (Figure 14E). Arrows in Figure 14E indicate overlapping P-H3 and DAPI fluorescence. These data support the hypothesis that the dormancy markers identified in Fig. 13B correlate with growth suppression in vitro and in vivo in short-term experiments in the CAM system. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 14E] See legend to Figure 14A. [Figure 15A]Figures 15A-15C show the evaluation of BMP7-F9 treatment in a mouse model of disease. Figure 15A is a schematic diagram of the in vivo experimental procedure used to evaluate the effect of BMP7-F9 on metastasis initiation. HEp3-GFP HNSCC tumors were allowed to grow to approximately 300 mm3 and then treated with 50 μg / kg of BMP7-F9 in a neoadjuvant setting until tumors reached approximately 600 mm3. Tumors were then surgically removed. One to two days after surgery, adjuvant treatment with BMP7-F9 was continued for an additional 3, 4, or 6 weeks. Animals were then euthanized, and lung DCC burden was scored using a fluorescent microscope. Figure 15B shows that BMP7 limits the occurrence of local and distant recurrence after tumor surgery. NSG mice were treated according to the protocol in Figure 15A for 3 and 6 weeks. At these time points, the percentage of local recurrence and DCC appearance were scored. In Figure 15C, mice were treated as in Figure 15A, but with adjuvant treatment for 4 weeks. After treatment, the number of GFP-positive cells in excised lungs was scored. This represents the magnitude of DCC burden in the lungs, and is significantly reduced by BMP7-F9 treatment. Note that the median DCC burden is 1 log lower, and that BMP-7 clearly cured 3 of 7 animals of DCC. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description Disclosed herein is a method for treating minimal residual cancer in a subject. One aspect of the present disclosure relates to a method for treating minimal residual cancer in a subject. The method includes contacting disseminated cancer cells (DCCs) in the subject with a bone morphogenetic protein 7 (BMP7) derivative protein. Contacting disseminated cancer cells (DCCs) in the subject with a bone morphogenetic protein 7 (BMP7) derivative protein induces or maintains dormancy in the contacted DCCs in the subject, thereby treating minimal residual cancer in the subject.

[0014] As used herein, the phrase "minimal residual cancer" includes a situation or condition in which there is no evidence of cancer in a subject by standard radiographic and histological standards, but the subject does in fact have residual cancer cells (i.e., DCCs) in the blood (as CTCs) or in the bone marrow or lymph nodes (as DTCs). Minimal residual cancer can occur after cancer treatment with chemotherapy, surgery, and / or radiation therapy. Standard radiographic and histological detection methods can include, for example, imaging tests (x-ray, ultrasound, MRI); blood or immunochemical tests for known tumor markers such as PSA or circulating tumor markers; and biopsy or cytological testing for known tumor markers to assess, for example, the number of tumor cells present or the relative rarity of such cells.

[0015] It is well known in the art that tumor cells can disseminate from primary tumor as CTC and DTC at early stage.In fact, DTC has been identified in subjects without evidence of disease after tumor surgery.In rare cases where cancer history does not preclude transplantation, even if donor is disease-free for 30 years, recipient develops donor-derived metastasis (MacKie et al., "Fatal Melanoma Transferred in a Donated Kidney 16 Years after Melanoma Surgery," N. Engl. J. Med. 348:567-568 (2003), the entire text of which is incorporated herein by reference).

[0016] Whole-genome sequencing of tumor phylogenies and metastases within individual patients suggests primary tumor-to-metastasis and metastasis-to-metastasis spread, supporting the inability of continuous / linear growth models to explain late (>10 years) recurrence in individual patients (Gundem et al., "The Evolutionary History of Lethal Metastatic Prostate Cancer," Nature 520:353-357 (2015) and Naxerova et al., "Using Tumor Phylogenetics to Identify the Roots of Metastasis in Humans," Nature Reviews Clinical Oncology 12:258-272 (2015), incorporated herein by reference in their entireties).

[0017] Single-cell CTC analysis has also demonstrated genetic linkages between CTCs and primary tumors (Ni et al., "Reproducible Copy Number Variation Patterns Among Single Circulating Tumor Cells of Lung Cancer Patients," PNAS 110(52):21083-88; Heitzer et al., "Complex Tumor Genomes Inferred from Single Circulating Tumor Cells by Array-CGH and Next-Generation Sequencing," Cancer. Res. 73:2965-75 (2013); and Lohr et al., "Whole-Exome Sequencing of Circulating Tumor Cells Provides a Window into Metastatic Prostate Cancer," Nature Biotech. 32:479-484 (2014), incorporated herein by reference in their entireties).

[0018] Furthermore, in humans, CTCs / DTCs do not correlate with the stage or size of the primary cancer (Krishnamurthy et al., "Detection of Minimal Residual Disease in Blood and Bone Marrow in Early Stage Breast Cancer," Cancer 116(14):3330-3337 (2010), incorporated herein by reference in its entirety). Instead, CTCs and DTCs are thought to retain the ability to form metastatic / recurrent disease. Specifically, detection of CTCs and DTCs has been shown to predict metastasis and recurrence of breast and prostate cancer (Braun et al., "A Pooled Analysis of Bone Marrow Micrometastasis in Breast Cancer," NEJM 353:793-802 (2005); Hayes et al., "Circulating Tumor Cells at Each Follow-up Time Point During Therapy of Metastatic Breast Cancer Patients Predict Progression-Free and Overall Survival," Clin. Cancer Res. 12(14):4218-4224 (2006); and de Bono et al., "Circulating Tumor Cells Predict Survival Benefit from Treatment in Metastatic Castration-Resistant Prostate Cancer," Clin. Cancer Res. 14:6302-6309 (2008), which are incorporated herein by reference in their entireties).

[0019] Metastases are thought to arise from reactivated DCCs, which are proliferative but dormant. Given that patients develop metastases many years after tumor removal, dormant DCCs may be a major cause of late cancer recurrence. As used herein, the term "dormancy" refers to the temporary cessation of mitosis and proliferation and is defined as cellular dormancy, in which intrinsic and / or extrinsic mechanisms drive single or small groups of DCCs to enter a quiescent state (reversible growth arrest). A second category of dormant lesions is defined by angiogenic dormancy, in which a constant tumor mass is maintained by a balance between dividing cells and cells that die due to poor vascularization. The third category is immune-mediated dormancy, in which the immune system maintains a constant proliferating tumor mass through persistent cytotoxic activity that continuously reduces the proliferating cancer cell population (see, e.g., Sosa et al., "Mechanisms of Disseminated Cancer Cell Dormancy: An Awakening Field," Nat. Rev. Cancer 14(9):611-622 (2014), incorporated herein by reference in its entirety). Dormant cells can arise from pre-existing primary tumors, secondary tumors, and / or pre-invasive lesions.

[0020] In one embodiment of the methods disclosed herein, the contacted DCCs are dormant cancer cells, i.e., the cancer cells are temporarily in mitotic / growth arrest or exhibiting senescence-like behavior.

[0021] DCCs can be detected in bone marrow aspirates by positive staining for EpCAM or CK8 / 18 after negative selection to eliminate hematopoietic cells. Cells may also be stained for dormancy markers to determine whether they are proliferating or dormant. The latter can be performed after fixation. To perform whole-genome or whole-transcriptome analysis, bone marrow-derived EpCAM-positive DCCs are isolated alive and processed for whole-genome or whole-transcriptome analysis (Guzvic et al., "Combined Genome and Transcriptome Analysis of Single Disseminated Cancer Cells from Bone Marrow of Prostate Cancer Patients Reveals Unexpected Transcriptomes," Cancer Res. 74(24):7383-94 (2014), incorporated herein by reference in its entirety).

[0022] The methods described herein may further include detecting the presence of DCCs in the subject prior to the contacting step. As shown in Table 1 below, dormant DCCs are identifiable because their phenotype is distinct from other cell types (Sosa et al., "Mechanisms of Disseminated Cancer Cell Dormancy: An Awakening Field," Nat. Rev. Cancer 14(9):611-622 (2014), incorporated herein by reference in its entirety).

[0023] (Table 1) DCC markers TIFF0007719135000001.tif210154

[0024] DTCs have been identified in the bone marrow of 13-72% of prostate cancer patients before surgery and in 20-57% of patients with no evidence of disease more than 5 years after surgery (Morgan et al., "Disseminated Tumor Cells in Prostate Cancer Patients after Radical Prostatectomy and without Evidence of Disease Predicts Biochemical Recurrence," Clin. Cancer Res. 15:677-683 (2009) and Weckermann et al., "Perioperative Activation of Disseminated Tumor Cells in Bone Marrow of Patients with Prostate Cancer," J. Clin. Oncol. 27(10):1549-56 (2009), incorporated herein by reference in their entireties). Detection of DTCs is a prognostic factor for recurrence in clinically dormant patients.

[0025] As used herein, the phrase "clinical dormancy" refers to a prolonged period of clinical disease-free time (eg, greater than 5 years) from removal of the primary tumor until disease recurrence. Clinical dormancy is common in prostate cancer, breast cancer, esophageal cancer, kidney cancer, thyroid cancer, B-cell lymphoma, and melanoma (Lam et al., “The Role of the Microenvironment - Dormant Prostate Disseminated Tumor Cells in the Bone Marrow,” Drug Discovery Today Technol. 11:41-47 (2014); Gelao et al., “Tumor Dormancy and Clinical Implications in Breast Cancer,” Cancer Medical Science 7:320 (2013); Ellis et al., “Detection and Isolation of Prostate Cancer Cells from Peripheral Blood and Bone Marrow,” Urology 61:277-281 (2003); Morgan et al., “Disseminated Tumor Cells in Prostate Cancer Patients after Radical Prostatectomy and without Evidence of Disease Predicts Biochemical Recurrence,” Clin. Cancer Res. 15:677-683 (2009); and Pfitzenmaier et al., "Telomerase Activity in Disseminated Prostate Cancer Cells," BJU Int. 97:1309-1313 (2006), incorporated herein by reference in their entireties), reside in distant organs such as bone, lymph nodes, liver, and lungs where they can remain dormant for long periods (e.g., more than 10 years) before being able to produce clinical metastases in some patients.

[0026] In some embodiments of practicing the methods described herein, the subject has been diagnosed with CTCs.

[0027] In some embodiments of practicing the methods described herein, the subject has been diagnosed with DTC and / or non-metastatic cancer.

[0028] As used herein, a "subject" refers to a patient, such as a cancer patient, and includes any animal, but is preferably a mammal. In one embodiment, the subject is a human subject. Suitable human subjects include, but are not limited to, children, adults, and geriatric subjects diagnosed with disseminated cancer cells and / or non-metastatic cancer.

[0029] In other embodiments, the subject may be a cow, sheep, pig, cat, horse, mouse, dog, rabbit, or the like.

[0030] When practicing the methods described herein, DCCs in a subject are contacted to induce or maintain dormancy of the DCCs, which refers to the establishment of a sustained non-proliferative state of the DCCs or the continuation of the non-proliferative state of the DCCs.

[0031] In one embodiment, minimal residual cancer is treated in a subject who has been diagnosed with cancer. For example, but not limited to, the subject has been diagnosed with one or more of breast cancer, multiple myeloma, lung cancer, non-small cell lung cancer, brain cancer, cervical cancer, mantle cell lymphoma, leukemia, hepatocellular carcinoma, prostate cancer, uveal and cutaneous melanoma, skin cancer, head and neck cancer, thyroid cancer, glioblastoma, neuroblastoma, and colorectal cancer.

[0032] Other cancers may also be suitable for treatment with the methods described herein.

[0033] In one embodiment, the subject is treated with minimal residual cancer related to or associated with breast cancer.The breast cancer can be selected from one or more of invasive breast cancer, ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and inflammatory breast cancer.

[0034] Breast cancer can be molecularly classified according to various molecular factors, including hormone receptor and human epidermal growth factor receptor 2 (HER2) status. The HER2 and basal-like groups are the major molecular subtypes identified in hormone receptor-negative breast cancer (Schnitt, "Classification and Prognosis of Invasive Breast Cancer: From Morphology to Molecular Taxonomy," Modern Pathology 23:S60-S64 (2010), incorporated herein by reference in its entirety). In one embodiment, breast cancer is HER2 + It's breast cancer.

[0035] In some embodiments of the methods described herein, the subject has undergone surgical removal to remove the tumor. For example, the subject may have undergone one or more of the following: mastectomy, prostatectomy, skin lesion removal, small bowel removal, gastrectomy, thoracotomy, adrenalectomy, appendectomy, colectomy, oophorectomy, thyroidectomy, hysterectomy, glossectomy, colon polypectomy, and colorectal removal.

[0036] In the method described herein, disseminated cancer cells (DCCs) in a subject are contacted with a bone morphogenetic protein 7 (BMP7) derivative protein. BMP7 is a member of the TGFβ superfamily and is secreted by bone marrow stromal osteoblasts, which may affect the DCC / DTC microenvironment. BMP7 plays a key role in the transformation of mesenchymal cells into bone and cartilage, and has also been shown to reversibly induce senescence in prostate cancer stem-like cells (Kobayashi et al., "Bone Morphogenetic Protein 7 in Dormancy and Metastasis of Prostate Cancer Stem-Like Cells in Bone," J. Exp. Med. 208(13):2641-55 (2011), incorporated herein by reference in its entirety). Pro-BMP7 is an intermediate between pre-BMP7 and mature BMP7, which is generated by proteolytic processing of the precursor protein, generating a subunit of the mature homodimer.

[0037] The human BMP7 protein is a secreted signaling molecule of the TGF-beta superfamily that was initially identified for its ability to induce bone formation but was later recognized as a multifunctional cytokine that mediates the proliferation and differentiation of many different cell types. Human BMP7 protein is expressed intracellularly as a 292 amino acid precursor protein, and the mature, biologically active BMP7 is generated by proteolytic removal of the signal peptide and propeptide. The amino acid sequence of wild-type human BMP7 protein, including the signal peptide (first 29 amino acids), prodomain, and mature peptide (bold), is shown below as SEQ ID NO:1: TIFF0007719135000002.tif49152. Those skilled in the art will appreciate that the signal peptide is removed by proteolytic cleavage to yield an intact prodomain / mature peptide, designated pro-BMP7.

[0038] Wild-type human mature BMP7 is a dimer of two 139 amino acid glycosylated disulfide-linked homodimerizing proteins of approximately 35 kDa. Each homodimerizing protein has the sequence shown in SEQ ID NO:2: It has the amino acid sequence shown in TIFF0007719135000003.tif16152.

[0039] Variants of the human BMP7 protein include variants of the human mature BMP7 of SEQ ID NO:2, which is represented by SEQ ID NO:3: The variants have specific amino acid changes as set forth in the consensus sequence set forth in TIFF0007719135000004.tif16152. Certain variants of the human mature BMP7 protein of the present disclosure have increased specific activity, improved solubility characteristics, improved bioavailability, decreased binding to endogenous circulating inhibitors, and / or decreased EBF activity compared to wild-type mature human BMP7 protein.

[0040] Preferred variants of the human BMP7 protein are selected from the group consisting of F93V / N110G; Y65G / I86L / T89A / N110G; Y65G / I86L / N110G / Y128F; Y65G / I86L / N110G / Y128W; Y65G / I86L / F93V / N110G / Y128W (BMP7-F9); Y65G / T89A / N110G / Y128F; Y65G / I86L / N110G; and Y65G / V114M (see Table 2 below).

[0041] Table 2. Exemplary variants of human BMP7 TIFF0007719135000005.tif111154TIFF0007719135000006.tif141154

[0042] In one embodiment, the variant of BMP7 is selected from the group consisting of Y65G / I86L / N110G / Y128W and Y65G / I86L / F93V / N110G / Y128W.

[0043] In one embodiment, the BMP7 derivative is a modified BMP7 variant of pro-BMP7. The modified variant of pro-BMP7 may include amino acid substitutions at amino acid positions corresponding to the BMP7 mature protein domain. The modified variant of pro-BMP7 may be processed to form a mature BMP7 derivative protein. A suitable pro-BMP7 variant containing a prodomain fused to the N-terminus of a human mature BMP7 protein variant is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16 shown in Table 3.

[0044] Table 3. Exemplary variants of human Pro-BMP7 TIFF0007719135000007.tif230151TIFF0007719135000008.tif103151

[0045] Suitable BMP7 derivative proteins for use in the methods described herein include variants of human pre-BMP7 (ie, SEQ ID NO:1).

[0046] In one aspect, a BMP7 derivative protein is a mature BMP7 protein that has increased biological activity (e.g., up to 50-fold or more biological activity) and biophysical properties (e.g., increased solubility and stability) compared to the mature wild-type BMP7 protein.

[0047] In another embodiment, the BMP7 derivative is BMP7-F9 (SEQ ID NO:8).

[0048] As used herein, a reference to a wild-type BMP7 protein or a variant thereof, including a reference to a SEQ ID NO, refers to a homodimer in which each monomeric subunit has the specified sequence. For example, a reference to BMP7-F9 (SEQ ID NO:8) refers to a homodimer in which each monomeric subunit has the sequence set forth in SEQ ID NO:8, and the subunits are linked by disulfide bonds.

[0049] In the functional assays described herein, treatment with or administration of a particular pro-BMP7 protein or variant thereof refers to treatment with or administration of a particular homodimer of mature BMP7, i.e., wild-type or variant thereof, generally in a non-covalent complex with the wild-type human prodomain.

[0050] According to the methods described herein, the contacting step can be carried out by administering a BMP7 derivative protein to the subject.

[0051] The effect of BMP7 on a subject may be dependent on BMP receptor 2 (BMPR2), and expression of this receptor has been shown to be inversely correlated with recurrence and bone metastasis in prostate cancer patients (Kobayashi et al., "Bone Morphogenetic Protein 7 in Dormancy and Metastasis of Prostate Cancer Stem-Like Cells in Bone," J. Exp. Med. 208(13):2641-55 (2011), the entire text of which is incorporated herein by reference). Thus, in one embodiment, the DCCs to be contacted in the subject are bone morphogenetic protein receptor-positive (BMPR2). + )

[0052] The methods described herein may further include administering to the subject a chemotherapeutic agent, an immunotherapeutic agent, an epigenetic agent, or ionizing radiation.

[0053] As used herein, the term "chemotherapeutic agent" refers to a synthetic, biological, or semi-synthetic compound that is not an enzyme and that kills or inhibits the growth of cancer cells but has little effect on non-cancerous cells. Any suitable chemotherapeutic agent can be used.

[0054] Suitable chemotherapeutic agents include, but are not limited to, anthracyclines, taxanes, kinase inhibitors, antibodies, fluoropyrimidines, and platinum drugs. Exemplary anthracyclines include, but are not limited to, doxorubicin, daunorubicin, epirubicin, mitoxantrone, and idarubicin. Exemplary taxanes include, but are not limited to, docetaxel and paclitaxel. Exemplary kinase inhibitors include, but are not limited to, lapatinib, imatinib mesylate, and gefitinib. Exemplary antibodies include, but are not limited to, alemtuzumab, gemtuzumab ozogamicin, rituximab, trastuzumab, and ibritumomab tiuxetan. Exemplary fluoropyrimidines include, but are not limited to, 5-fluoruracil, capecitabine, tegafur, tegafur-uracil, floxuridine, 5-fluorodeoxyuridine, and S-1. Exemplary platinum drugs include, but are not limited to, cisplatin, carboplatin, oxaliplatin, and nedaplatin.

[0055] Additional suitable chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, thiotepa, hexamethylmelamine, busulfan, carmustine, lomustine, semustine, streptozocin, decarbazine, estramustine, streptozocin, and temozolomide), vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine), podophyllotoxins (e.g., etoposide and teniposide), antibiotics (e.g., bleomycin, dactinomycin, mitomycin, and valrubicrin), and camptothecin analogs (e.g., irinotecan or topotecan).

[0056] In some embodiments, the chemotherapeutic agent is an anti-HER2 chemotherapeutic agent selected from trastuzumab (Herceptin®) and lapatinib (Tykerb®). Trastuzumab is a monoclonal antibody that targets the HER2 / neu receptor on cancer cells. Lapatinib is a tyrosine kinase inhibitor that targets the epidermal growth factor receptor (EGFR) and HER2.

[0057] As used herein, the term "immunotherapeutic agent" refers to an agent that can induce or enhance an immune response in a subject. In the context of cancer, immunotherapeutic agents stimulate the immune system to more effectively target cancerous cells. Suitable immunotherapeutic agents may be selected from immune checkpoint inhibitors, interferons, and tumor vaccines.

[0058] Immune checkpoint inhibitors are compounds that inhibit the binding of immune checkpoints. Exemplary immune checkpoint modulators include PD-1 inhibitors (e.g., pembrolizumab and nivolumab), PD-L1 inhibitors (e.g., atezolizumab, avelumab, and durvalumab), and CTLA-4 inhibitors (e.g., ipilimumab).

[0059] Interferons ("IFNs") are a family of cytokines that protect against disease by directly affecting target cells and by activating the immune response. IFNs can be produced by and act on both tumor cells and immune cells. Type I IFNs include IFNα proteins, IFNβ, IFNε, IFNκ, and IFNω. Type I IFNs are known to mediate anti-neoplastic effects against several types of malignancies (Moschos et al., "Interferons in the Treatment of Solid Tumors," Cancer Treat. Res. 126:207-241 (2005), incorporated herein by reference in its entirety).

[0060] As used herein, the term "tumor vaccine" refers to a composition that stimulates an immune response against tumors or cancerous cells in a subject. Tumor vaccines typically consist of a cancer-related substance or cell (antigen) source, which can be autologous (derived from the subject) or allogeneic (derived from another person) to the subject, along with other components (e.g., adjuvants) to further stimulate and boost the immune response to the antigen. Tumor vaccines can stimulate the subject's immune system to produce antibodies against one or several specific antigens and / or to produce killer T cells that attack cancer cells bearing those antigens.

[0061] As used herein, the term "epigenetic agent" refers to an agent that alters the epigenetic state (e.g., methylation state) of a cell's DNA upon contact with or administration of such agent.

[0062] Suitable epigenetic agents may be selected from, for example, histone deacetylase ("HDAC") inhibitors, 5-azacytidine, retinoic acid, arsenic trioxide, Zeste2 enhancer polycomb repressive complex 2 subunit ("EZH2") inhibitors, bromodomain ("BRD") inhibitors, and derivatives thereof.

[0063] Exemplary HDAC inhibitors include, but are not limited to, trichostatin A, trapoxin B, benzamide, phenylbutyrate, valproic acid, vorinostat, belinostat, LAQ824, panobinostat, entinostat, CI994, and mocetinostat.

[0064] Exemplary EZH2 inhibitors include, but are not limited to, 3-deazaneplanocin A (DZNep), EPZ005687, GSK126, EI1, UNC1999, and EPZ-6438 (Kim et al., "Targeting EZH2 in Cancer," Nat. Med. 22(2):128-134 (2016), incorporated herein by reference in its entirety).

[0065] Exemplary bromodomain inhibitors include, but are not limited to, JQ1, I-BET151 / 762, PF-1, and RVX-208 (Wadhwa et al., "Bromodomain Inhibitor Review: Bromodomain and Extra-terminal Family Protein Inhibitors as a Potential New Therapy in Central Nervous System Tumors," Cureus 8(5):e620 (2016), incorporated herein by reference in its entirety).

[0066] Further exemplary epigenetic agents include DNA methyltransferase (DNMT) inhibitors, such as, but not limited to, azacytidine and decitabine.

[0067] The DCC / DTC microenvironment plays a crucial role in enhancing dormancy. Nuclear receptor subfamily 2 group F member 1 (NR2F1) is a nuclear hormone receptor and transcription factor, and is a key node in the transcription factor network that constitutes the tumor cell dormancy signature. Estrogen receptor-positive (ER) +) When applied to gene expression profiles of breast cancer patients, this signature has been shown to predict a longer metastasis-free interval (Kim et al., “Dormancy Signatures and Metastasis in Estrogen Receptor Positive and Negative Breast Cancer,” PloS One 7:e35569 (2012), incorporated herein by reference in its entirety). This dormancy signature has also been found in dormant DTCs in prostate cancer patients who had been asymptomatic for 7 to 18 years (Sosa et al., "NR2F1 Controls Tumor Cell Dormancy via SOX9- and RARbeta-Driven Quiescence Programmes," Nat. Commun. 6:6170 (2015) and Chery et al., "Characterization of Single Disseminated Prostate Cancer Cells Reveals Tumor Cell Heterogeneity and Identifies Dormancy Associated Pathways," Oncotarget 5:9939-51 (2014), incorporated herein by reference in their entireties), demonstrating its relevance to human disease.

[0068] NR2F1 has been shown to be upregulated after tumor surgery in head and neck squamous cell carcinoma (HNSCC) patient-derived xenograft (PDX) models and induce dormancy of local and distant residual tumor cells (Sosa, "Dormancy Programs as Emerging Antimetastasis Therapeutic Alternatives," Mol. Cell. Oncol. 3(1):e1029062 (2016), incorporated herein by reference in its entirety). The plasticity of NR2F1 expression suggests that epigenomic changes in residual tumor cells can be controlled by external and internal signals and may determine the fate of DCCs. NR2F1 has been shown to restrict reprogramming of induced pluripotent stem cells (iPS cells), likely by regulating chromatin reprogramming (Onder et al., "Chromatin Modifying Enzymes as Modulators of Reprogramming," Nature 483(7391):598-602 (2012), incorporated herein by reference in its entirety). NR2F1 is also key to maintaining overall repressive chromatin in dormant tumor cells while simultaneously allowing active chromatin states at the promoters of specific dormant genes, including their own promoters (Sosa et al., "NR2F1 Controls Tumor Cell Dormancy via SOX9- and RARbeta-Driven Quiescence Programmes," Nat. Commun. 6:6170 (2015), incorporated herein by reference in its entirety), highlighting the existence of a coordinated epigenetic program regulated by NR2F1 and microenvironmental cues that leads to tumor cell dormancy. In one embodiment, the DTC is NR2F1 + is.

[0069] DCCs have been shown to exhibit high levels of PERK pathway activation, a mediator of the ISR (Bragado et al., “Microenvironments Dictating Tumor Cell Dormancy,” Recent Results Cancer Res. 195:25-39 (2012); Sosa et al., “Regulation of Tumor Cell Dormancy by Tissue Microenvironments and Autophagy,” Adv. Exp. Med. Biol. 734:73-89 (2013); Goswami et al., “The Phosphoinositide 3-Kinase / Akt1 / Par-4 Axis: A Cancer-Selective Therapeutic Target,” Cancer Res. 66(6):2889-92 (2006); and Schewe et al., “ATF6alpha-Rheb-mTOR Signaling Promotes Survival of Dormant Tumor Cells in vivo,” PNAS 105(30):10519-24 (2008), incorporated herein by reference in its entirety. ISR signaling and EIF2α phosphorylation by PERK results in a global decrease in translation and gene-specific translation, oxidative stress, and increased ROS production, protein degradation, RNA degradation, autophagy, and lipid biosynthesis, which may aid in tumor cell survival.

[0070] Another aspect relates to a method of treating minimal residual cancer in a subject, the method comprising contacting disseminated cancer cells (DCCs) in the subject with a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor selected from LY2, LY3, and LY4, thereby eradicating the DCCs in the subject and treating the minimal residual cancer in the subject.

[0071] In one embodiment, the DCC is phospho-PERK active. Thus, the method can further include contacting the DCC in the subject with a PERK inhibitor, a MEK inhibitor, a CDK4 / 6 inhibitor, or any combination thereof.

[0072] In one embodiment of the methods described herein, the contacting step can be carried out by administering a PERK inhibitor to the subject.

[0073] In one embodiment, the PERK inhibitor is represented by formula (I): TIFF0007719135000009.tif37128, or a pharmaceutically acceptable salt thereof; In the formula, R is Selected from the group consisting of TIFF0007719135000010.tif28128; X is CH or N; R 1 is hydrogen or halogen (e.g., fluoro); R 2 is C1-C3 alkyl.

[0074] In a further embodiment, the PERK inhibitor is represented by formula (Ia): TIFF0007719135000011.tif42128, or a pharmaceutically acceptable salt thereof; In the formula, R is Selected from the group consisting of TIFF0007719135000012.tif30128; X is CH or N; R 1 is hydrogen or halogen (e.g., fluoro); R 2 is C1-C3 alkyl.

[0075] When the inhibitor is a compound of formula (I) or formula (Ia), R is It could also be TIFF0007719135000013.tif30128.

[0076] As used herein, the term "alkyl" refers to alkyl groups having about 1 to about 6 carbon atoms, or 1 to about 3 carbon atoms (alternatively, the number of carbon atoms is "C") in the chain. n ~C n ", where n is a range of the number of carbon atoms. Branched means that one or more lower alkyl groups, such as methyl, ethyl, or propyl, are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, and i-propyl.

[0077] The term "halogen" means fluoro, chloro, bromo, or iodo. In one aspect, the halogen is fluoro.

[0078] The term "compound" and equivalent expressions refer to compounds described herein, and includes, where the context permits, prodrugs, pharmaceutically acceptable salts, oxides, and solvates, such as hydrates.

[0079] The compounds described herein may contain one or more asymmetric centers and may therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms. Each chiral center may be defined in terms of absolute stereochemistry as (R)- or (S)-. The present invention is intended to encompass all such possible isomers, as well as mixtures thereof, including racemic and optically pure forms. Optically active (R)- and (S)-, (-)- and (+)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. All tautomeric forms are also intended to be encompassed.

[0080] Reference to a "compound" is intended to include salts, solvates, oxides, and inclusion complexes of that compound, as well as any stereoisomeric form, or mixtures comprising the compound in any such form, in any proportion. Thus, in some embodiments, the compounds described herein are provided in the form of a salt, including within the context of pharmaceutical compositions, methods of treatment, and the compounds themselves.

[0081] The term "solvate" refers to a compound in the solid state in which molecules of a suitable solvent are included in the crystal lattice. A suitable solvent for therapeutic administration is one in which the administered dose is physiologically acceptable. Examples of suitable solvents for therapeutic administration are ethanol and water. When the solvent is water, the solvate is called a hydrate. Solvates are generally formed by dissolving the compound in a suitable solvent and isolating the solvate by cooling or using an antisolvent. Solvates are typically dried or azeotroped under ambient conditions.

[0082] Inclusion complexes are described in Remington, The Science and Practice of Pharmacy, 19th Ed. 1:176-177 (1995), which is incorporated herein by reference in its entirety. The most widely used inclusion complexes are those involving cyclodextrins, and all natural and synthetic cyclodextrin complexes are specifically encompassed by the present invention.

[0083] The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases.

[0084] The term "pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for use in contact with cells of humans and lower animals, without significant toxicity, irritation, allergic response, etc., and commensurate with a reasonable benefit / risk ratio.

[0085] Suitable PERK inhibitors may be selected from LY2, LY3, LY4, and combinations thereof (see Table 4 below). The PERK inhibitor may be a pharmaceutically acceptable salt of LY2, LY3, and / or LY4.

[0086] Table 4. Exemplary PERK inhibitors TIFF0007719135000014.tif135154

[0087] In some embodiments, the contacting step is carried out with a PERK inhibitor that does not inhibit EIF2AK1, EIF2AK2, or EIF2AK4.

[0088] In one embodiment, the PERK inhibitor does not inhibit AXL. In this embodiment, the PERK inhibitor is selected from LY3 and LY4.

[0089] In another embodiment, the PERK inhibitor does not inhibit Flt3, MNK2, or NTRK. In this embodiment, the PERK inhibitor is LY4.

[0090] In one embodiment, the contacting step is carried out by administering a MEK inhibitor to the subject. Exemplary MEK inhibitors are known in the art, and include, for example, PD184352, PD318088, PD98059, PD334581, and RDEA119 / BAY 869766 (see, e.g., Iverson et al., "RDEA119 / BAY 869766: A Potent, Selective, Allosteric Inhibitor of MEK1 / 2 for the Treatment of Cancer," Cancer Res. 69(17):6839-47 (2009), the entire contents of which are incorporated herein by reference).

[0091] In another embodiment, the contacting step is carried out by administering a CDK4 / 6 inhibitor to the subject. Exemplary CDK4 / 6 inhibitors are well known in the art and include, for example, abemaciclib (LY2835219), palbociclib (PD0332991), and ribociclib (LEE011).

[0092] In one aspect, the method can further include selecting a subject without evidence of disease prior to said contacting step, e.g., the subject can be in cancer remission prior to said contacting step.

[0093] When carrying out the methods described herein, minimal residual cancer in a subject is treated. Such treatment may include, but is not limited to, administering to a subject in need of minimal residual cancer treatment one or more compounds that are effective in treating the subject's condition (i.e., cancer or minimal residual cancer).

[0094] In one aspect, the therapeutic methods of the present disclosure are carried out under conditions effective to induce disseminated tumor cell ("DTC") dormancy and / or induce dormant DTC death.

[0095] When practicing the therapeutic methods of the present disclosure, administering a compound to a subject can include administering a therapeutically effective amount of a pharmaceutical composition containing the compound (i.e., the BMP7 derivative protein and PERK inhibitor of the present disclosure), where a therapeutically effective amount refers to an amount of compound effective to treat the described condition and / or disorder in a subject. Such amounts generally vary depending on several factors familiar to those skilled in the art. Such factors include, but are not limited to, the specific subject, as well as the subject's age, weight, height, general health, and medical history; the specific compound used, as well as the carrier in which it is formulated, and the selected route of administration; the length or duration of the treatment; and the nature and severity of the condition being treated.

[0096] Administering typically includes administering a pharmaceutically acceptable dosage form, which refers to dosage forms of the compounds described herein, including, for example, liquid preparations such as tablets, dragees, powders, elixirs, syrups, suspensions, sprays, inhalable tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories, as well as injectable liquid preparations, such as liposomal preparations. Techniques and formulations are extensively described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference in its entirety.

[0097] When carrying out the therapeutic method of the present disclosure, the drug (i.e., the BMP7 derivative protein and PERK inhibitor of the present disclosure) can be contained in any suitable amount in any suitable carrier substance. The drug can be present in an amount of up to 99% by weight based on the total weight of the composition. The composition can be provided in a dosage form suitable for oral, parenteral (e.g., intravenous, intramuscular), rectal, dermal, nasal, vaginal, inhalation, skin (patch), or intraocular administration routes. Thus, the composition can be in the form of, for example, a tablet, capsule, pill, powder, granule, suspension, emulsion, solution, gel such as a hydrogel, paste, ointment, cream, plaster, drench, osmotic delivery device, suppository, enema, injection solution, implant, spray, or aerosol.

[0098] The pharmaceutical compositions of the present disclosure may be formulated to release the active drug substantially immediately upon administration or at any predetermined time or period after administration.

[0099] Sustained-release formulations include: (i) formulations that produce a substantially constant drug concentration in the body over an extended period of time; (ii) formulations that produce a substantially constant drug concentration in the body over an extended period of time after a predetermined lag time; (iii) formulations that prolong the action of a drug over a predetermined period of time by maintaining a relatively constant effective drug level in the body while minimizing undesirable side effects associated with fluctuations in plasma levels of the active drug; (iv) formulations that localize the action of a drug, for example, by placing a sustained-release composition spatially adjacent to or within an affected cell, tissue, or organ; and (v) formulations that target the action of a drug by using a carrier or chemical derivative to deliver the drug to a specific target cell type.

[0100] Drug administration in the form of sustained-release formulations is beneficial because the drug (i) has a narrow therapeutic index (i.e., the difference between the plasma concentration that produces adverse side effects or toxic reactions and the plasma concentration that produces a therapeutic effect is small; generally, the therapeutic index (TI) is less than the median lethal dose (LD) 50 ) versus the median effective dose (ED 50 ) ratio); (ii) a narrow absorption window in the gastrointestinal tract; or (iii) a very short biological half-life, which requires multiple daily doses to maintain therapeutic plasma concentrations.

[0101] In order to obtain sustained release, the release rate of the drug of interest exceeds the metabolic rate of the drug, any of several strategies can be implemented.Sustained release can be obtained by appropriate selection of various formulation parameters and components, for example, various types of sustained release compositions and coatings.Therefore, drug is formulated with suitable excipients into pharmaceutical composition, and this composition releases the drug in a controlled manner when administered (single or multiple unit tablet or capsule composition, oily solution, suspension, emulsion, microcapsule, microsphere, nanoparticle, patch and liposome).

[0102] Thus, administration according to the methods of the present disclosure may be carried out orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, intracavity or intravesical instillation, intraocularly, intraarterially, intralesionally, or by mucosal application. The compounds may be administered alone or together with a suitable pharmaceutical carrier and may be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.

[0103] The drugs (i.e., the BMP7 derivative proteins and PERK inhibitors of the present disclosure) may be orally administered, for example, using an inert diluent or an edible carrier that can be digested, or may be enclosed in hard or soft shell capsules, compressed into tablets, or incorporated directly with dietary food. For therapeutic oral administration, the drugs may be combined with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.001% of the active compound. The percentage of the compound in these compositions may, of course, be varied and may conveniently comprise from about 0.01% to about 10% of the weight of the unit. The amount of active compound in such therapeutic compositions is adjusted to provide a suitable dosage. In one embodiment, the compositions are prepared so that an oral dosage unit contains from about 1 μg to 1 g of the active compound.

[0104] Tablets, capsules, etc. may also contain binders such as tragacanth, acacia, cornstarch, or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as cornstarch, potato starch, alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

[0105] Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar, or both. In addition to the active ingredient, the syrup may contain sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes, and flavorings such as cherry or orange flavor.

[0106] The therapeutic substance may also be administered parenterally. Solutions or suspensions can be prepared in water, appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in oils such as glycerol, liquid polyethylene glycols, and mixtures thereof. Exemplary oils include those derived from petroleum, animals, plants, or synthetic sources, such as peanut oil, soybean oil, or mineral oil. Generally, water, saline, aqueous solutions of dextrose and related sugars, and glycols such as propylene glycol, hyaluronan and its derivatives, carboxymethylcellulose, and other soluble polysaccharide derivatives, or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms if not produced sterilely.

[0107] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The forms must be sterile and fluid to the extent that they can be easily syringably drawn. They must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0108] The therapeutic substance can be administered directly to the airways in the form of an aerosol. When used as an aerosol, a solution or suspension of the therapeutic substance can be packaged in an aerosol pressurized container together with a suitable propellant, such as a hydrocarbon propellant such as propane, butane, or isobutane, and a common adjuvant. The therapeutic substance can also be administered in a non-pressurized manner, such as with a nebulizer or atomizer.

[0109] In one embodiment, administration may increase the amount of detectable dormant DCC in a subject by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more.

[0110] In another embodiment, administration may reduce the amount of detectable DCC in a subject by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more.

[0111] In the context of this disclosure, "treating" means maintaining a state in which there is no evidence of symptomatic disease (eg, cancer) in a subject.

[0112] In one aspect, the term " treating " or " treatment " particularly refers to the eradication of minimal residual cancer in subject.The term treatment includes the induction of dormancy of DCC.The term treatment also includes the eradication of dormant DCC in subject.The term treatment also includes the reduction of the amount or number of detectable dormant DCC in subject.

[0113] Another aspect of the present disclosure relates to a method of treating minimal residual cancer in a subject, the method comprising contacting disseminated cancer cells (DCCs) in the subject with a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor selected from LY2, LY3, and LY4, thereby eradicating the DCCs in the subject and treating the minimal residual cancer in the subject.

[0114] As noted above, the methods of the present disclosure are suitable for treating minimal residual cancer in subjects diagnosed with any one or more of breast cancer, multiple myeloma, lung cancer, non-small cell lung cancer, brain cancer, cervical cancer, mantle cell lymphoma, leukemia, hepatocellular carcinoma, prostate cancer, melanoma, skin cancer, head and neck cancer, thyroid cancer, glioblastoma, neuroblastoma, colorectal cancer, and other cancers.

[0115] The cancer can be a breast cancer selected from invasive breast cancer, ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and inflammatory breast cancer.

[0116] In one aspect, the breast cancer is HER2 + It's breast cancer.

[0117] In another embodiment, the subject has been diagnosed with disseminated tumor cells and / or non-metastatic cancer.

[0118] As noted above, the methods of the present disclosure may further include administering to the subject a chemotherapeutic agent, an immunotherapeutic agent, an epigenetic agent, or ionizing radiation.

[0119] When a chemotherapeutic agent is administered to the subject, the chemotherapeutic agent can be an anti-HER2 chemotherapeutic agent selected from trastuzumab (Herceptin®) and lapatinib (Tykerb®). In another embodiment, the chemotherapeutic agent can be selected from an anthracycline, a taxane, a kinase inhibitor, an antibody, a fluoropyrimidine, and a platinum drug.

[0120] When an immunotherapeutic agent is administered to the subject, the immunotherapeutic agent is selected from an immune checkpoint inhibitor, interferon, or a tumor vaccine.

[0121] When an epigenetic agent is administered to the subject, the epigenetic agent may be selected from a histone deacetylase (HDAC) inhibitor, 5-azacytidine, retinoic acid, arsenic trioxide, a Zeste2 enhancer polycomb repressive complex 2 subunit ("EZH2") inhibitor, a bromodomain (BRD) inhibitor, and derivatives thereof.

[0122] The contacting step can be carried out by administering to the subject a PERK inhibitor. Suitable PERK inhibitors are described in detail above and include, but are not limited to, LY2, LY3, and LY4.

[0123] In one embodiment, the method further comprises detecting the presence of DTC in the subject prior to said contacting step. As described in more detail above, DTC can be a protein that is specifically NR2F1 + , phospho-PERK activity, and / or BMPR + It could be.

[0124] The method may further comprise contacting DCC / DTCs in the subject with a BMP7 derivative protein. In one embodiment, contacting DCC / DTCs in the subject with a BMP7 derivative protein is carried out by administering the BMP7 derivative protein to the subject. Suitable BMP7 derivative proteins are described above. In one embodiment, the BMP7 derivative protein is BMP7-F9.

[0125] In one embodiment, the PERK inhibitor does not inhibit EIF2AK1, EIF2AK2 or EIF2AK4.

[0126] As noted above, the subject may be a mammal, preferably a human.

[0127] In one aspect, the method can further include selecting a subject without evidence of disease prior to said contacting step, e.g., the subject can be in cancer remission prior to said contacting step.

[0128] Yet another aspect of the present disclosure relates to a method of treating late-stage cancer in a subject, the method comprising contacting disseminated cancer cells (DCCs) in the subject with a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor selected from LY2, LY3, and LY4, thereby eradicating the DTCs in the subject and treating minimal residual cancer in the subject.

[0129] As used herein, the term "late stage cancer" refers to stage II cancer, stage III cancer, and / or stage IV cancer, or any cancer that has metastasized. It will be understood that the "late stage" of a cancer disease state can be determined by a physician.

[0130] As detailed above, the subject may have been diagnosed with breast cancer, multiple myeloma, lung cancer, non-small cell lung cancer, brain cancer, cervical cancer, mantle cell lymphoma, leukemia, hepatocellular carcinoma, prostate cancer, melanoma, skin cancer, head and neck cancer, thyroid cancer, glioblastoma, neuroblastoma, or colorectal cancer.

[0131] In one embodiment, the cancer is a breast cancer selected from invasive breast cancer, ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and inflammatory breast cancer. + It could be breast cancer.

[0132] The method may further comprise administering to the subject a chemotherapeutic agent, an immunotherapeutic agent, an epigenetic agent, or ionizing radiation. In one embodiment, the chemotherapeutic agent is an anti-HER2 chemotherapeutic agent selected from trastuzumab (Herceptin®) and lapatinib (Tykerb®). In another embodiment, the chemotherapeutic agent is selected from an anthracycline, a taxane, a kinase inhibitor, an antibody, a fluoropyrimidine, and a platinum drug. The immunotherapeutic agent may be selected from an immune checkpoint inhibitor, an interferon, or a tumor vaccine. The epigenetic agent may be selected from a histone deacetylase (HDAC) inhibitor, 5-azacytidine, retinoic acid, arsenic trioxide, a Zeste2 enhancer polycomb repressive complex 2 subunit (EZH2) inhibitor, a bromodomain (BRD) inhibitor, and derivatives thereof.

[0133] In one embodiment, the contacting step is carried out by administering a PERK inhibitor to the subject.

[0134] The method may further comprise detecting the presence of DCC / DTC in the subject prior to the contacting step.

[0135] As mentioned above, the DTC is NR2F1 + Or it may be phospho-PERK activity. [Example]

[0136] Materials and Methods for Examples 1 - 2-6 Reagents, cell culture, and treatments: EGF was obtained from PeproTech (Rocky Hill, NJ) and used at 100 ng / ml. Thapsigargin was obtained from Sigma (St. Louis, MO) and used at 2 nM. The ZR75.1-H2B-Dendra2 cell line was generated by stable transfection of the H2B-Dendra2 plasmid (Gurskaya et al., "Engineering of a Monomeric Green-to-Red Photoactivatable Fluorescent Protein Induced by Blue Light," Nat. Biotechnol. 24:461-465 (2006), incorporated herein by reference in its entirety). For 3D culture, MCF10A-HER2, SKBR3, and ZR75.1-H2B-Dendra2 cells were plated and grown in growth factor-reduced Matrigel (Corning, NY) as previously described (Avivar-Valderas et al., "Regulation of Autophagy during ECM Detachment is Linked to a Selective Inhibition of mTORC1 by PERK," Oncogene 32(41):4932-40 (2013), incorporated herein by reference in its entirety). When referring to "low density," 3,500 cells were seeded per 8 wells, and when referring to "high density," 20,000 cells were seeded per 8 wells. Treatment with vehicle (DMSO) or LY4 (2 μM) was changed every 24 hours for 2D and every 48 hours for 3D culture.

[0137] Mice, Tumor Growth, and Tissue Processing: The FVB / N-Tg(MMTVneu) mouse strain was obtained from Jackson Laboratories (Sacramento, CA). These mice express an inactivated neu (HER2) form under the transcriptional control of the mouse mammary tumor virus promoter / enhancer. Females were pregnant once and weaned and then withheld from lactation for at least two weeks before use. Females aged 24–32 weeks were intraperitoneally injected with vehicle (90% corn oil, 10% ethanol) or LY4 (50 mpk) daily for two weeks. For combined treatment, 24–32 week-old females were treated with abemaciclib (50 mpk) by oral gavage daily for four weeks, after which treatment with LY4 was initiated as described above. Tumor volume was calculated using the formula (D × d) 2 ) / 2, where D is the largest diameter and d is the smallest diameter. For CTC counting, animals were anesthetized and exsanguinated via cardiac puncture. Mammary glands, lungs, and tumors were collected and fixed overnight in 10% buffered formalin before being embedded in paraffin. Bone marrow from two hind limbs was flushed with a 26-gauge needle and further processed by Ficoll density gradient centrifugation. To detect CTCs and DTCs in the bone marrow, mature hematopoietic cells in the tissue were depleted by anti-mouse antibody-labeled magnetic bead separation (Miltenyi Biotec, San Diego, CA) and then fixed in formalin for 20 minutes at 4°C.

[0138] Mammary gland whole-mount staining: Mammary glands fixed in 10% buffered formalin were incubated in Carmine Alum stain (0.2% carmine, 0.5% potassium aluminum sulfate) (Sigma, St. Louis, MO) for 2 days, then dehydrated and transferred to methyl salicylate solution before imaging under a stereomicroscope.

[0139] IHC and IF: IHC and IF of paraffin-embedded sections were performed as previously described (Avivar-Valderas et al., "Regulation of Autophagy during ECM Detachment is Linked to a Selective Inhibition of mTORC1 by PERK," Oncogene 32(41):4932-40 (2013), incorporated herein by reference in its entirety). Briefly, slides were dewaxed and serially rehydrated. Heat-induced antigen retrieval was performed in citrate buffer (10 mM, pH 6), EDTA buffer (1 mM, pH 8), or Tris / EDTA (pH 9). Slides were further permeabilized with 0.1% Triton™-X100, blocked, and incubated with primary antibodies at 1:50–1:200 dilutions overnight at 4°C. For IHC, additional steps included internal peroxidase and avidin / biotin quenching before primary antibody incubation.The primary antibodies used were anti-cytokeratin 8 / 18 (Progen, Heidelberg, Germany), smooth muscle actin-Cy3 (Sigma, St. Louis, MO), P-PERK (T980) (Tenkerian et al., “mTORC2 Balances AKT Activation and eIF2alpha Serine 51 Phosphorylation to Promote Survival Under Stress,” Mol. Cancer Res. 13:1377-1388 (2015), incorporated herein by reference in its entirety), P-EIF2A, cleaved caspase 3, P-H3 (S10), P-HER2 (Y1221 / 1222) (Cell signaling, Danvers, MA), P-Rb (S249 / T252) (Santa Clara, Dallas, TX). The antibodies were: Santa Cruz), HER2 (Abcam, Cambridge, MA), HER2 (Millipore, Darmstadt, Germany), Ki67 (eBioscience and Abcam), cytokeratin cocktail (C11 and ck7, Abcam; AE1 and AE3, Millipore), and GADD34 (Santa Cruz). Slides were then incubated in secondary antibodies (Life Technologies, Norwalk, CT) and mounted. For IHC, sections were treated with the VectaStain ABC Elite kit (Vector Laboratories, Burlingame, CA) and a DAB substrate kit for peroxidase labeling (Vector Laboratories) and mounted in VectaMount medium (Vector Laboratories). For IF, sections were mounted in ProLong Gold Antifade aqueous medium (Thermo Fisher, Waltham, MA).

[0140] For immunohistofluorescence, cytospins of fixed cells (100,000–200,000 cells / cytospin) were prepared onto poly-prep slides by cytocentrifugation at 500 rpm for 3 minutes. After permeabilization, the staining protocol was performed as described below. For 3D culture staining, acini were fixed in 4% PFA for 20 minutes at 4°C, permeabilized with 0.5% Triton™-X100 in PBS for 20 minutes at room temperature, washed with PBS-glycine, and then blocked with 10% normal goat serum for 1 hour at 37°C before immunofluorescence staining.

[0141] Scoring of P-HER2 levels is illustrated in Figure 10A. CK8 / 18 and SMA in the ducts were scored in 20 low-magnification fields per animal, with CK8 / 18 expression assessed as negative (0), low (1), or high (2), and SMA as well, and the sum of the two scores was used to determine the final score (0–4).

[0142] Microscopy: Images were taken with a Nikon Eclipse TS100 microscope, a Leica DM5500, or a confocal Leica SP5 multiphoton microscope.

[0143] TUNEL in situ cell death detection: Apoptosis levels were assessed using the In situ Cell Death Detection Kit AP (Roche, Basel, Switzerland). Tumor paraffin sections were dewaxed, rehydrated, and permeabilized with 0.2% Triton™-X100 in phosphate-buffered saline (PBS) for 8 minutes. The slides were then washed and blocked in 20% normal goat serum for 1 hour at 37°C. The TUNEL reaction mixture was then added and left at 37°C for 1 hour. The reaction was stopped by incubation with Buffer I (0.3 M sodium chloride, 30 mM sodium citrate). The slides were then incubated with anti-fluorescein-AP antibody for 30 minutes at 37°C. After three washes with Tris-buffered saline (TBS), the slides were incubated in alkaline phosphatase substrate in 0.1% TWEEN™-20 for 20 minutes at room temperature. Finally, the slides were mounted using aqueous mounting medium. The percentage of TUNEL-positive cells was calculated using Image J software (NIH).

[0144] Immunoblot analysis: Cells were lysed in RIPA buffer, and protein analysis was performed by immunoblot as previously described (Ranganathan et al., "Functional Coupling of p38-Induced Up-Regulation of BiP and Activation of RNA-Dependent Protein Kinase-Like Endoplasmic Reticulum Kinase to Drug Resistance of Dormant Carcinoma Cells," Cancer Res. 66:1702-1711 (2006), incorporated herein by reference in its entirety). Membranes were blotted with additional antibodies against P-PERK (T982) (Tenkerian et al., "mTORC2 Balances AKT Activation and eIF2alpha Serine 51 Phosphorylation to Promote Survival Under Stress," Mol. Cancer Res. 13:1377-1388 (2015), incorporated herein by reference in its entirety), PERK (Santa Cruz, Dallas, TX), P-EGFR (Y1148), EGFR, P-AKT (S473), P-S6 (S235 / 236) (Cell signaling, Danvers, MA), GAPDH (Millipore, Darmstadt, Germany), and β-tubulin (Abcam, Cambridge, MA). To induce ER stress, MCF10A-HER2 cells were plated on low-adhesion plates for 24 hours and then harvested.

[0145] Cell surface biotinylation and endocytosis assay: For cell surface biotinylation, the Pierce Cell Surface Protein Isolation Kit was used according to the manufacturer's instructions with minor modifications. Briefly, MCF10A-HER2 cells were serum- and EGF-starved and treated with + / - LY4 for 24 hours, followed by stimulation with + / - EGF (100 ng / ml) for 20 minutes. Cells were then washed with ice-cold PBS, and surface proteins were biotinylated for 30 minutes at 4°C. After quenching, cells were harvested and lysed using RIPA buffer. Protein lysates were incubated with NeutrAvidin agarose beads, and bound proteins were released by incubation with SDS-PAGE sample buffer containing DTT (50 mM). For the endocytosis assay (Cihil et al., "The Cell-Based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins," J. Vis. Exp. e50867 (2013), incorporated herein by reference in its entirety), cells were treated similarly, except that cell surface proteins were biotinylated before EGF treatment. After incubation with + / - EGF (100 ng / ml) for 20 min at 37°C (to induce endocytosis), cells were washed with ice-cold PBS and incubated for 30 min with detachment buffer (to remove cell surface biotinylation: 75 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 50 mM glutathione, and 80 mM NaOH, pH 8.6). To control for detachment efficiency, cells were detached without incubation at 37°C (t=0). Cell lysates were prepared and treated as described above to isolate biotinylated proteins.

[0146] Single-cell target gene expression analysis: Primary tumors from 28-30 week-old MMTV-neu females were digested with collagenase to form single-cell suspensions. Lungs from 15-30 week-old MMTV-neu females were digested with collagenase to form single-cell suspensions and resuspended in FACS buffer. Cells were then stained with anti-HER2-PE, anti-CD45-APC, and DAPI, and the HER2+ / CD45- cell population was sorted using a BDFACSAria sorter. Sorted cells were resuspended in medium at a concentration of 312,500 cells / ml, and 80 μl was mixed with 20 μl of suspension reagent (C1 Fluidigm). Single cells were isolated using a C1 Single-cell Preamp IFC 10-17 μm. Preamplification was performed using the Ambion Single Cell-to-CT qRT-PCR Kit and a 20× TaqMan Gene Expression FAM-MGB assay. The resulting cDNA was further diluted in 1 / 3 C1 DNA dilution reagent and used for gene expression analysis using a 96.96 IFC (Fluidigm), a Juno System controller for high-throughput qPCR, and a Biomark HD. TaqMan Fast Advanced Master Mix was used for qPCR reactions. Analysis was performed using Fluidigm Real-Time PCR Analysis Software and the Clustergrammer web-based tool (Fernandez et al., "Clustergrammer, A Web-Based Heatmap Visualization and Analysis Tool for High-Dimensional Biological Data," Sci. Data 4:1-12 (2017), incorporated herein by reference in its entirety) to generate a hierarchical clustering heatmap.

[0147] Biochemical Assay: Recombinant human EIF2AK3 (PERK) catalytic domain (amino acids 536–1116; Cat# PV5107), GFP-eIF2α (Cat# PV4809) substrate, and terbium-labeled phospho-eIF2α antibody (Cat# PR8956B) were purchased from Invitrogen (Carlsbad, CA). The HIS-SUMO-GCN2 catalytic domain (amino acids 584–1019) was expressed in Escherichia coli (E. coli) and purified. TR-FRET kinase assays were performed in the presence or absence of inhibitors in a reaction buffer consisting of 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1.0 mM EGTA, and 0.01% Brij-35, and 100–200 nM GFP-eIF2α substrate. The PERK assay was performed using 62.5 ng / ml of enzyme and 1.5 μM ATP (K m, app Approximately 1.5 μM), and the GCN2 assay was performed with 3 nM enzyme and 90 μM ATP (K m, app The reaction mixture contained approximately 200 μM of EDTA. After the addition of the test compound, the reaction was initiated by the addition of enzyme and incubated at room temperature for 45 minutes. The reaction was stopped by adding EDTA to a final concentration of 10 mM, and terbium-labeled phospho-eIF2α antibody was added to a final concentration of 2 nM and incubated for 90 minutes. The resulting fluorescence was monitored using an EnVison® Multilabel reader (PerkinElmer, Waltham, MA). The TR-FRET ratio and the resulting IC 50 Values were determined from fitted inhibition curves. Biochemical specificity profiling was performed at Cerep (Redmond, WA) and DiscoverX (San Diego, CA).

[0148] Cell-based TR-FRET assay: Briefly, GripTite™ 293 cells (Invitrogen) expressing GFP-eIF2α were seeded at 10,000 cells / well in 384-well plates and allowed to adhere overnight. Cells were pretreated with test compounds for 1 hour. Tunicamycin (1 μM) was added to induce PERK activity, and the plates were incubated at 37°C for 2 hours. The culture medium was removed, and cells were lysed in a buffer consisting of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 5 mM NaF, protease inhibitors (Sigma Cat# P8340), phosphatase inhibitors (Sigma Cat# P2850), and 2 nM terbium-labeled anti-phospho-eIF2 antibody (Invitrogen Cat# PM4312I). The cell lysates were incubated in the dark for 2 hours at room temperature, and fluorescence was observed using an EnVison® Multilabel reader (PerkinElmer, Waltham, MA). TR-FRET ratios and resulting IC50 values were determined from fitted inhibition curves using uninduced (100% inhibition) and induced (0% inhibition) wells as controls.

[0149] ATF4-luc assay: 293 cells were transduced with ATF4-luc-expressing lentivirus (SABiosciences, Frederick, MD) and selected in growth medium containing 1 μg / ml puromycin. To determine the effect of compounds on ER stress-induced ATF4 activity, 293-ATF4-luc cells were seeded at 15,000 cells / well in poly-D-lysine-coated 96-well plates and allowed to adhere overnight. Cells were then pretreated with test compounds for 30 minutes. ER stress was induced by the addition of tunicamycin (2 μM), and the plates were incubated at 37°C for 6 hours. The culture medium was then aspirated, and the cells were lysed in passive lysis buffer (Promega Cat# E194A) for 5 minutes on a plate shaker. Luciferase activity was monitored using luciferase assay reagent (Promega Cat# E1501) on a Wallac 1420 Victor2™ Multilabel Counter (PerkinElmer, Waltham, MA), and IC values were calculated from the fitted inhibition curves obtained using uninduced (100% inhibition) and induced (0% inhibition) wells as controls. 50 value was determined.

[0150] Cell viability assay: Hela, HT-1080, and Bx-PC-3 cell proliferation was monitored in 96-well plates in the absence or presence of PERK inhibitors for 48, 72, or 96 hours, respectively. Cell viability was determined using CellTiter-Glo® reagent (Promega, Madison, WI). IC values were calculated from fitted inhibition curves using untreated (0% inhibition) and 20 μM staurosporine-treated (100% inhibition) wells as controls. 50 value was determined.

[0151] Statistical analysis: All points represent independent biological samples, error bars represent standard deviation, and statistical significance was determined by the Mann-Whitney test using Graph Pad Prism Software.

[0152] Example 2 - Silent HER2 +DTCs exhibit an ER stress response PERK pathway activation has been shown to be a key effector of UPR-induced growth arrest and survival associated with a dormant phenotype (Brewer et al., "PERK Mediates Cell-Cycle Exit During the Mammalian Unfolded Protein Response," Proc. Natl. Acad. Sci. USA 97:12625-30 (2000); Ranganathan et al., "Dual Function of Pancreatic Endoplasmic Reticulum Kinase in Tumor Cell Growth Arrest and Survival," Cancer Res. 68:3260-3268 (2008); and Ranganathan et al., "Functional Coupling of p38-Induced Up-Regulation of BiP and Activation of RNA-Dependent Protein Kinase-Like Endoplasmic Reticulum Kinase to Drug Resistance of Dormant Carcinoma Cells," Cancer Res. 66:1702-1711 (2006), each of which is incorporated herein by reference in its entirety).In MMTV-HER2 animals, a high percentage of mice develop lung metastases, which can be initiated by early or late DCC (Guy et al., "Expression of the Neu Protooncogene in the Mammary Epithelium of Transgenic Mice Induces Metastatic Disease," Proc. Nat'l. Acad. Sci. USA 89:10578-10582 (1992); Husemann et al., "Systemic Spread Is an Early Step in Breast Cancer," Cancer Cell 13:58-68 (2008); Harper et al., "Mechanism of Early Dissemination and Metastasis in Her2. + Dormant DCCs exhibit loss of E-cadherin and expression of Twist1 (Harper et al., “Mechanism of Early Dissemination and Metastasis in Her2”), whereas dormant DCCs exhibit loss of E-cadherin and expression of Twist1 (Hosseini et al., “Early Dissemination Seeds Metastasis in Breast Cancer,” Nature 540:588-592 (2016); Hosseini et al., “Early Dissemination Seeds Metastasis in Breast Cancer,” Nature 540:552-558 (2016), which are incorporated herein by reference in their entireties). +In a pancreatic cancer model, E-cadherin-negative DCCs were also found to be quiescent and exhibit upregulation of the PERK-inducible gene CHOP (Pommier et al., "Unresolved Endoplasmic Reticulum Stress Engenders Immune-Resistant, Latent Pancreatic Cancer Metastases," Science 360(6394):eaao4908 (2018), incorporated herein by reference in its entirety). To assess whether this same correlation between PERK pathway activation levels and cell cycle arrest exists in the MMTV-HER2 spontaneous metastasis model, we used two different approaches: high-resolution imaging using immunofluorescence (IF) and single-cell resolution gene expression analysis of DCCs and metastases. IF was performed on MMTV-HER2 lung tissue sections from animals with large tumors and therefore dormant and proliferative DCCs (Harper et al., “Mechanism of Early Dissemination and Metastasis in Her2 + (Mammary Cancer,” Nature 540:588-592 (2016), incorporated herein by reference in its entirety). Tissues were then co-stained to detect HER2, Ki67 (as a proliferation marker), and GADD34 (or PPP1r15A)-positive DCCs. GADD34 is a PERK-inducible stress gene responsible for a programmed shift from translational repression (by eIF2α phosphorylation) to stress-induced gene expression (Novoa et al., “Stress-Induced Gene Expression Requires Programmed Recovery from Translational Repression,” EMBO J. 22:1180-7 (2003), incorporated herein by reference in its entirety). Image analysis revealed that DCCs with a low proliferation index (ki67 low ) HER2 +Metastatic lesions or DCCs exhibited high levels of ER stress, as indicated by high levels of GADD34 expression (Fig. 1A, upper panel and graph). On the other hand, highly proliferative DCCs or lesions showed very low levels of GADD34 staining (Fig. 1A, lower panel and graph). The two markers, Ki67 and GADD34, were inversely correlated in 100% of cells, indicating that GADD34 detection was associated with the UPR. high These results support the idea that CTCT can be a biomarker for quiescent DCC and metastatic disease.

[0153] We next assessed whether these correlations also hold true for human breast metastatic lesions by examining 17 breast cancer metastases of different subtypes and sources (lymph node, lung, and liver) (Table 5). Breast cancer metastases were stained for cytokeratin to identify metastatic lesions, Ki67, and GADD34. Compared to the mouse model, advanced human metastatic lesions displayed more heterogeneous staining patterns for both markers between patients and between different regions of the same lesion. However, regardless of metastatic type, an inverse correlation between proliferation levels (Ki67) and ER stress activation levels (GADD34) was observed (Figure 1B). This analysis supports the findings in the mouse model and the role of GADD34 in the UPR at metastatic sites. high / It has been demonstrated that it can be useful in identifying quiescent tumor cells.

[0154] Table 5. Human breast cancer metastasis samples TIFF0007719135000015.tif139149TIFF0007719135000016.tif144149*High (H); Low (L); Intermediate (I).

[0155] Markers of proliferation, quiescence, dormancy, and ER stress present in metastatic cells were assessed by performing targeted gene expression analysis of single cells in DCCs, micrometastases, and macrometastases within the lungs of MMTV-HER2 mice. Lungs from MMTV-HER2 females were processed into single-cell suspensions, and HER2 + / CD45 -The cells were sorted (Figure 2A). Then, the sorted cells were processed for single cell isolation, lysis, RT, and pre-amplification using C1 (Fluidigm) technology, as shown in Figure 2A. This pipeline was used to generate high-confidence (HER2) + This allowed for the isolation and processing of 255 single DCCs and 90 primary tumor cells and their corresponding pools with high quality (single-cell IF and molecular confirmation). Next, high-throughput qPCR was used to analyze the expression of ER stress genes, cell cycle genes (both activators and inhibitors), and dormancy genes (Kim et al., "Dormancy Signatures and Metastasis in Estrogen Receptor Positive and Negative Breast Cancer," PloS One 7:e35569 (2012), incorporated herein by reference in its entirety; B'chir et al., "The eIF2α / ATF4 Pathway is Essential for Stress-Induced Autophagy Gene Expression," Nucleic Acids Res. 41:7683-99 (2013); Harper et al., "Mechanism of Early Dissemination and Metastasis in Her2 +"Mammary Cancer," Nature 540:588-592 (2016), each of which is incorporated herein by reference in its entirety (Figure 2B). Single-cell resolution gene expression of DCCs revealed the presence of a cell population (Figure 1C, group 1, approximately 19% of DTCs) that exhibited concomitant and strong upregulation of all ER stress genes examined (including PERK itself) (box surrounding Fam123b-Ddit3), along with negative regulators of cell proliferation such as Rb1 and TP53, and the CDK inhibitors p21, p27, p16, and p15 (box surrounding Cdkn2a-Rb1) (Figure 1C). In these cells, we also observed increased expression of dormancy genes such as NR2F1, DEC2 (Bhlhe41), TWIST1, CDH5, STAT3, and COL4a5 (Kim et al., "Dormancy Signatures and Metastasis in Estrogen Receptor Positive and Negative Regulators of Cell Proliferation and Metastasis"). Negative Breast Cancer,” PloS One 7:e35569 (2012) and Harper et al., “Mechanism of Early Dissemination and Metastasis in Her2 +Mammary Cancer,” Nature 540:588-592 (2016), each incorporated herein by reference in its entirety) (box surrounding Nr2f1-Ccnd1). Another group of DCCs, Group 2 (22%), also showed high levels of ER stress gene expression along with p21. Group 3 (6%) showed fewer ER stress, cell cycle inhibitor, and dormancy genes, suggesting that they may be cells transitioning out of dormancy or in a slow cycling mode. In total, approximately 40% of DCCs showed high to moderate levels of ER stress gene expression along with cell cycle inhibitor or dormancy genes. This is in line with the percentage of dormant DCCs detected by phospho-histone H3 and phospho-Rb detection in MMTV-HER2 animals that progressed to progression (Harper et al., “Mechanism of Early Dissemination and Metastasis in Her2 + ("Mammary Cancer," Nature 540:588-592 (2016), incorporated herein by reference in its entirety). Collectively, this data demonstrates that even in animals with detectable metastases, approximately 40% of DCCs exhibit high expression of cell cycle inhibitors. Importantly, in this model, this dormant DCC subpopulation exhibits an unresolved UPR accompanied by marked activation of PERK pathway genes.

[0156] Example 3 - PERK inhibition eradicates quiescent DCCs in bone marrow and lung, thereby suppressing lung metastasis Given these results, we evaluated the effects of selective PERK inhibitors on dormant DCC fate and metastasis formation. LY2, LY3, and LY4 (LY series inhibitors) have been described as potent and selective PERK inhibitors with suitable drug-like properties to support in vivo studies (Pytel et al., “PERK Is a Haploinsufficient Tumor Suppressor: Gene Dose Determines Tumor-Suppressive Versus Tumor Promoting Properties of PERK in Melanoma,” PLoS Genet. 12:1–22 (2016), incorporated herein by reference in its entirety). The LY series inhibitors were tested in in vitro kinase assays (using eIF2α as a substrate) and cell-based assays to examine eIF2α phosphorylation and downstream ATF4 output (Table 6). All three inhibitors showed comparable or superior potency compared to GSK2656157 (Axten et al., “Discovery of GSK2656157: An Optimized PERK Inhibitor Selected for Preclinical Development,” ACS Med. Chem. Lett. 4:964-968 (2013), incorporated herein by reference in its entirety); effectively reduced P-PERK (P-T980) levels and its downstream target ATF4 in HER2-expressing MCF10A cells (Figures 2C and 3A); and sensitized these same cells to low-dose thapsigargin treatment, thus demonstrating how these PERK inhibitors selectively affect ER stress adaptation (Figure 2D).

[0157] Biochemical enzyme assays (Figure 2E) showed that LY4 showed the highest specificity, exhibiting no secondary kinase targets at concentrations below 15 μM, whereas LY2, LY3, and GSK2656157 exhibited several secondary targets at concentrations below 5 μM and even at 1 μM. xScanMAX™ kinase profiling (Table 6) confirmed that LY4 exhibited high selectivity over other inhibitors; even at very high concentrations (20 μM), LY4 inhibited only 20 of a total of 456 kinases by >50% compared to 80 for GSK2656157, or >60% inhibition in 8 compared to 58. None of these secondary targets were other known eIF2α kinases, EIF2AK1 (also known as HRI), EIF2AK2 (also known as PKR), and EIF2AK4 (also known as GCN2) (Table 7), indicating that the measured activity against eIF2α was highly specific for PERK inhibition.

[0158] Table 6. Enzyme- and cell-based IC of different PERK inhibitors 50 Value and kinase selectivity TIFF0007719135000017.tif62154 a PERK biochemical assay using purified eIF2a as substrate. b Cell-based assay of tunicamycin-induced eIF2a phosphorylation in 293 cells. c Cell-based assay of tunicamycin-induced ATF4-Luc activity in 293 cells. d GCN2 biochemical assay using purified eIF2a as substrate. e DiscoverR based on binding data using displacement of active site probes x scanMAX™ Kinase Profiling, tests 456 kinases. f Atkins et al., “Characterization of a Novel PERK Kinase Inhibitor with Antitumor and Antiangiogenic Activity,” Cancer Res. 73(6):1993-2002 (2013), incorporated herein by reference in its entirety.

[0159] Table 7. Comparison of in vitro inhibition of other eiF2α kinases TIFF0007719135000018.tif47155 a DiscoverR based on binding data using displacement of active site probes x scanMAX™ Kinase Profiling, tests 456 kinases. b Atkins et al., “Characterization of a Novel PERK Kinase Inhibitor with Antitumor and Antiangiogenic Activity,” Cancer Res. 73(6):1993-2002 (2013), incorporated herein by reference in its entirety.

[0160] Single-parous MMTV-HER2 female mice aged 24–32 weeks were treated with vehicle or LY4 (50 mpk) intraperitoneally daily for 2 weeks. Mammary glands, lungs, pancreas, bone marrow, and tumors were collected for further analysis. LY4 was well tolerated and did not significantly alter body weight, consistent with recent studies showing no effect on blood glucose levels or pancreatic function (Pytel et al., “PERK Is a Haploinsufficient Tumor Suppressor: Gene Dose Determines Tumor-Suppressive Versus Tumor-Promoting Properties of PERK in Melanoma,” PLoS Genet. 12:1–22 (2016), incorporated herein by reference in its entirety). The inhibitor had no significant effect on bone marrow cell homeostasis or peripheral blood leukocyte counts, as evidenced by no effect on total cell counts in MMTV-HER2 females (Figure 2F).

[0161] PERK inhibition resulted in a significant decrease in P-PERK and P-eIF2α levels in ductal and pancreatic tissues (although only partially, especially in pancreatic islets) (Figure 3B). We concluded that systemic delivery of LY4 effectively inhibits PERK activation and eIF2α phosphorylation. PERK inhibition did not completely deplete PERK activity, and thus mice may have been able to control pancreatic function and glucose levels (Yu et al., "Type I Interferons Mediate Pancreatic Toxicities of PERK Inhibition," Proc. Natl. Acad. Sci. 112:15420-15425 (2015), incorporated herein by reference in its entirety).

[0162] A high percentage of MMTV-HER2 animals develop lung metastases, which may initiate early in the progression of the disease (Guy et al., “Expression of the Neu Protooncogene in the Mammary Epithelium of Transgenic Mice Induces Metastatic Disease,” Proc. Nat'l. Acad. Sci. USA 89:10578-10582 (1992); Husemann et al., “Systemic Spread Is an Early Step in Breast Cancer,” Cancer Cell 13:58-68 (2008); Harper et al., “Mechanism of Early Dissemination and Metastasis in Her2 +Mammary Cancer,” Nature 540:588-592 (2016), Hosseini et al., “Early Dissemination Seeds Metastasis in Breast Cancer,” Nature 540:552-558 (2016); Linde et al., “Macrophages Orchestrate Breast Cancer Early Dissemination and Metastasis,” Nat. Commun. 9:21 (2018), the entire contents of which are incorporated herein by reference).

[0163] Therefore, the effect of the LY4 PERK inhibitor on metastatic disease was observed in animals with small tumors and / or large, palpable tumors. All vehicle-treated animals showed detectable metastases in H&E-stained sections. Lesions showing more than 100 cells were generally also positive for proliferation markers and were classified as macrometastases (Figure 1A). Quantification of macrometastases per animal (five non-consecutive lung sections) revealed that after only 2 weeks of treatment, LY4 reduced the number and incidence of macrometastases without affecting the area of these metastases (Figure 4A) (Figure 3C). This suggested that PERK inhibition may act at an earlier stage of metastasis rather than by reducing established macrometastases. Therefore, we next evaluated whether LY4 treatment could affect tumor cell intravasation from the primary site or the transition from solitary DCCs to micrometastases (containing 2–100 cells). HER2 in blood samples +Direct detection of circulating tumor cells (CTCs) showed no significant difference between vehicle- and LY4-treated animals (Figure 4B), indicating that LY4 does not significantly affect tumor cell intravasation. On the other hand, IHC detection of micrometastases and single DCCs by HER2 detection revealed a significant reduction in the number of micrometastases in LY4-treated females (Figure 3D). More than 80% of single DCCs in the lung were P-Rb negative, indicating that they were largely out of the cell cycle and dormant. This finding recapitulates that of a previous study (Harper et al., "Mechanism of Early Dissemination and Metastasis in Her2"). + (2016), incorporated herein by reference in its entirety.) Significantly, LY4 significantly reduced the number of nonproliferating (P-Rb-negative) single DCCs, which are typically associated with blood vessels, in lung sections without affecting the number of P-Rb-positive solitary DTCs (Figure 3E) or micrometastases (Figure 4C). Importantly, LY4 significantly reduced the number of DCCs found in the bone marrow (Figure 3F). In the bone marrow, DCCs are found at a high prevalence and remain dormant, yet do not develop any metastases (Bragado et al., "TGF-Beta2 Dictates Disseminated Tumor Cell Fate in Target Organs Through TGF-Beta-RIII and P38Alpha / Beta Signaling," Nat. Cell. Biol. 15:1351-1361 (2013); Husemann et al., "Systemic Spread Is an Early Step in Breast Cancer," Cancer Cell 13:58-68 (2008); and Harper et al., “Mechanism of Early Dissemination and Metastasis in Her2 +(2016), each of which is incorporated herein by reference in its entirety.) These results demonstrate that PERK inhibition selectively targets non-proliferating, dormant DCCs that exhibit active PERK and UPR signaling.

[0164] To further test whether LY4 treatment can selectively target the survival of human DCCs outside the cell cycle, we modeled this biology in 3D cultures of human ZR75.1 HER2 cells stably expressing a photoconvertible fluorescent protein (Dendra2) fused to histone H2B. + We performed long-term label-retention assays using cells because H2B-containing nucleosomes in quiescent cells turn over slowly (Wilson et al., “Hematopoietic Stem Cells Reversibly Switch From Dormancy to Self-Renewal During Homeostasis and Repair,” Cell 135:1118-1129 (2008), incorporated herein by reference in its entirety). After approximately 1 minute of exposure to 405 nm light, the H2B-DENDRA2 protein converts from green to red fluorescence, resulting in double-positive green and red cells. Cells that revert back to green divide and dilute the H2B-DENDRA2-RED molecule, while quiescent cells remain H2B-DENDRA2 GREEN and RED (Gurskaya et al., "Engineering of a Monomeric Green-to-Red Photoactivatable Fluorescent Protein Induced by Blue Light," Nat. Biotechnol. 24:461-465 (2006), incorporated herein by reference in its entirety). Macrometastasis-mimicking cells seeded at high density (clusters) or single DCC-mimicking cells seeded at low density (single cells) in a 3D Matrigel matrix were photoconverted (100%) (Figure 4D). After 8 days, only 35% of the high-density cells were H2B-DENDRA2-RED positive. In contrast, low-density ZR75.1 HER2+ Sixty-five percent of the cells were H2B-DENDRA2-RED positive (Figure 4E), mimicking the quiescent state of single DCCs (Bragado et al., "TGF-Beta2 Dictates Disseminated Tumor Cell Fate in Target Organs Through TGF-Beta-RIII and P38Alpha / Beta Signaling," Nat. Cell. Biol. 15:1351-1361 (2013), incorporated herein by reference in its entirety). Treatment with the PERK inhibitor LY4 significantly reduced the proliferation of densely seeded ZR75.1 HER2 cells. + However, the inactive single ZR75.1 HER2 + The cells were eradicated, consistent with the in vivo DCC results (Figure 3G). These data demonstrate that in the context of single tumor cells, intrinsic and / or ECM-dependent signals lead to a reliance on PERK signaling, and that LY4 indeed selectively targets slow-cycling or nonproliferating DCCs that subsequently reactivate to generate metastases.

[0165] Example 4 – PERK inhibition interferes with early and late HER2-induced mammary tumor progression Quiescent UPR most associated with diapause high Following the demonstration of PERK dependency in DCCs, tumor lesions were evaluated. HER2-induced progression was found to be genetically dependent on the PERK kinase in the MMTV-HER2 model (Bobrovnikova-Marjon et al., "PERK-Dependent Regulation of Lipogenesis During Mouse Mammary Gland Development and Adipocyte Differentiation," Proc. Nat'l. Acad. Sci. USA 105:16314-16319 (2008), incorporated herein by reference in its entirety), and HER2 +Tumors have been shown to be sensitive to proteotoxicity and ERAD-dependent (Singh et al., "HER2-mTOR Signaling-Driven Breast Cancer Cells Require ER-Associated Degradation to Survive," Sci. Signal. 8:ra52 (2015), incorporated herein by reference in its entirety). Furthermore, analysis of the cBIO database (Cerami et al., "The cBio Cancer Genomics Portal: An Open Platform for Exploring Multidimensional Cancer Genomics Data," Cancer Discovery 2:401-404 (2012), incorporated herein by reference in its entirety) revealed that approximately 14% of HER2-amplified human breast tumors exhibited upregulation of PERK mRNA (Figure 5A). Therefore, we investigated whether LY4 affected HER2-induced mammary tumor progression in primary lesions where various stages of progression could be analyzed, from hyperplastic mammary glands to DCIS and invasive carcinoma (Lu et al., "Mechanism of Inhibition of MMTV-neu and MMTV-wnt1 Induced Mammary Oncogenesis by RARalpha Agonist AM580," Oncogene 29(25):3665-76 (2010); Muller et al., "Single-Step Induction of Mammary Adenocarcinoma in Transgenic Mice Bearing the Activated c-neu Oncogene," Cell 54(1):105-115 (1988); Harper et al., "Mechanism of Early Dissemination and Metastasis in Her2 +Mammary Cancer,” Nature 540:588-592 (2016); Hosseini et al., “Early Dissemination Seeds Metastasis in Breast Cancer,” Nature 540:552-558 (2016), the entire contents of which are incorporated herein by reference).

[0166] Analysis of the mammary glands of 24-week-old, single-parous females revealed that vehicle-treated MMTV-HER2 animals exhibited ducts with dense secondary and tertiary branching (Figure 6A, left panel), and histological analysis revealed numerous glandular hyperplastic lesions (Figure 6A, right panel, black arrows). In contrast, LY4-treated animals exhibited a "normalized" ductal structure with fewer branches, similar to the mammary tree of non-transgenic, normal FVB mice (Figure 3B). LY4-treated animals also exhibited a significant increase in the number of hollow ducts, accounting for more than 60% of the total structure compared with approximately 20% in control females (Figure 6B and Figure 5C). The number of obstructive hyperplasia and DCIS-like lesions was also reduced to less than half of that in vehicle-treated animals. In contrast, LY4-treated animals exhibited a "normalized" ductal structure with less branching, similar to the mammary tree of non-transgenic, normal FVB mice (Figure 3B). LY4-treated animals also exhibited a significant increase in the number of hollow ducts, accounting for more than 60% of the total structure, compared with approximately 20% in control females (Figure 6B and Figure 5C). The number of obstructive hyperplasia and DCIS-like lesions was also reduced to less than half of that in vehicle-treated animals. + Hyperplastic lesions in these animals showed varying degrees of luminal differentiation, as assessed by heterogeneous cytokeratin 8 / 18 expression levels (Fig. 6C, upper panel). Myoepithelial cells (detected as positive for smooth muscle actin, SMA), which are evenly spaced in the ducts of normal FVB animals, were heterogeneously distributed in the hyperplasias of vehicle-treated MMTV-HER2 mice. In contrast, LY4-treated MMTV-HER2 animals showed increased cytokeratin 8 / 18 expression in the luminal layer, which often surrounds empty lumens, and in a continuous layer of outer myoepithelial cells (Fig. 6C, lower panel and graph). These data indicate that LY4 treatment results in the "normalization" of early cancer lesions, likely by restoring the differentiation program.

[0167] The animals ranged in volume from 30 to 200 mm 3 tumors (two tumors >200 mm 3The tumors were treated with LY4 for 2 weeks (Fig. 7A). In the vehicle-treated group, tumors grew steadily (Fig. 8A), reaching 10 times their original volume within 2 weeks (Fig. 7B, upper graph). In contrast, LY4-treated tumors showed a reduced growth rate (Fig. 8A), with some tumors remaining in a state of complete cell growth arrest (defined as tumor volume doubling within 2 weeks; 43% in LY4-treated tumors vs. 7% in control tumors) (Fig. 7B, lower graph). Some tumors (25%) even regressed within the 2-week treatment window (Fig. 7C). This resulted in a significant reduction in the median final tumor volume (Fig. 8B). Although proliferation levels (P-histone H3 IHC) were not different between vehicle- and LY4-treated tumors (Fig. 7D), TUNEL staining of tumor sections demonstrated a significant increase in DNA fragmentation levels in LY4-treated animals (Fig. 8C). Thus, in overt primary lesions, LY4 treatment resulted in pre-formed HER2 + Context-dependent adaptations were demonstrated to induce tumor apoptosis and promote PERK function during tumor progression.

[0168] Treatment of HER2-overexpressing (MCF10A-HER2 or ZR75.1) or HER2-amplified (SKBR3) human cancer cells in 3D Matrigel acinar cultures with LY4 (Figure 8D and Figure 7E) showed that treatment with vehicle or LY4 (2 μM) for 10 days significantly increased apoptosis levels (cleaved caspase-3) in these organoids, particularly in the inner cell mass that had lost contact with the ECM (Figure 8D). As in vivo, no significant changes in proliferation levels were observed, as detected by phospho-histone H3 levels (Figure 7F). Primary MMTV-HER2 + We conclude that PERK is required for HER2-induced changes in ductal epithelial architecture. + In human cancer cells and mouse tumors, HER2 is dependent on PERK for survival.

[0169] Example 5 - Optimal HER2 phosphorylation, localization, and AKT and ERK activation require PERK signaling HER2 +Because tumors are sensitive to proteotoxicity (Singh et al., "HER2-mTOR Signaling-Driven Breast Cancer Cells Require ER-Associated Degradation to Survive," Sci. Signal. 8:ra52 (2015), incorporated herein by reference in its entirety), we evaluated whether PERK inhibitors could affect optimal HER2 activity by increasing ER client protein load. Detection of HER2 phosphorylation at residues Y1221 / 1222 within tumors revealed that the P-HER2-positive area reported in other studies (DiGiovanna et al., "Active Signaling by Neu in Transgenic Mice," Oncogene 17:1877-1884 (1998), incorporated herein by reference in its entirety) overlapped with P-PERK and P-eIF2α staining (Figure 9A). This finding indicated colocalization of PERK and HER2 pathway activation. Similarly, single-cell targeted gene expression profiling of primary tumor cells also revealed a population of primary tumor cells (approximately 25%) with high levels of ER stress gene expression (Figure 9B), which may correspond to the population exhibiting P-HER2 activation. Importantly, when tumor P-HER2 levels were scored considering both the area and intensity of staining (Figure 10A), LY4-treated tumors were found to exhibit significantly lower levels of P-HER2 than control animals (Figure 9C).HER2 signals as a homodimer or heterodimer with EGFR and HER3 (Moasser MM, "The Oncogene HER2: Its Signaling and Transforming Functions and its Role in Human Cancer Pathogenesis," Oncogene 26(45):6469-87 (2007) and Negro et al., "Essential Roles of Her2 / erbB2 in Cardiac Development and Function," Recent Prog. Horm. Res. 59:1-12 (2014), each of which is incorporated herein by reference in its entirety). In vitro treatment of MCF10A-HER2 cells starved and treated with EGF (100 ng / ml, 15 min) in the presence or absence of LY4 (2 μM) revealed that the PERK inhibitor reduced both basal and EGF-induced levels of P-EGFR and P-HER2, as well as down-regulation of the survival pathways P-AKT, P-S6, and P-ERK1 / 2 (Figure 9D and graph, and Figure 10B). Under these conditions, no apparent effect on total HER2 levels or heterodimerization with EGFR was observed, as determined by surface biotinylation and coimmunoprecipitation. Because LY4 has no direct inhibitory effect on the active sites of any HER family members, AKT, or S6 kinases (Table 8), this effect may be due to an indirect PERK inhibitory effect on HER2 signaling.Unlike other HER family members, HER2 is known to remain in the cell membrane even after ligand binding and dimerization (Hommelgaard et al., "Association with Membrane Protrusions Makes ErbB2 an Internalization-Resistant Receptor," Mol Biol Cell. 15(4):1557-67 (2004); Bertelsen et al., "The Mysterious Ways of ErbB2 / HER2 Trafficking," Membranes (Basel) 4:424-446 (2014), each of which is incorporated herein by reference in its entirety). To test whether LY4 can interfere with the HER2 receptor activation mechanism, a surface biotinylation assay was performed to measure the presence of receptors on the cell surface, and reversible surface biotinylation was measured to measure receptor endocytosis (Cihil et al., "The Cell-Based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins," J. Vis. Exp. e50867 (2013), incorporated herein by reference in its entirety). The data showed that LY4 treatment reduced the amount of P-HER2 and total HER2 on the cell surface (Figures 9E and 10C), and concomitantly increased the endocytosis of phospho-HER2 and total HER2 (Figure 9F). This data, together with that of Singh et al., "HER2-mTOR Signaling-Driven Breast Cancer Cells Require ER-Associated Degradation to Survive," Sci. Signal. 8:ra52 (2015) (incorporated herein by reference in its entirety), suggests that PERK signaling and proper UPR function are required for maintaining proper HER2 downstream signaling by influencing optimal receptor localization and activation.

[0170] Table 8. Direct inhibitory activity of LY4 on the HER family signaling pathway TIFF0007719135000019.tif191155 1 DiscoverR based on binding data using displacement of active site probes x scanMAX™ Kinase Profiling, tests 456 kinases.

[0171] Example 6 - Sequential combination of CDK inhibitor followed by PERK inhibition enhances the anti-metastatic effect of LY4 We have established the efficacy of pharmacological PERK inhibition on primary lesions and, crucially, that LY4 can inhibit metastasis by eradicating dormant DCCs. Given this information, we hope to use clinically available drugs that mimic dormancy to induce UPR in dormant cancer cells. high The basic principle behind this is that UPR is a ubiquitous factor. high This finding suggests that DCCs expressed higher levels of CDK inhibitors (Fig. 1C). Therefore, we next asked whether pre-treating animals with the CDK4 / 6 inhibitor abemaciclib (50 mpk, 4 weeks) would further enhance the anti-metastatic effect of LY4 by increasing the pool of quiescent DCCs above basal 50% (Fig. 11A). Indeed, pre-treatment of MMTV-HER2 females with abemaciclib alone significantly increased GADD34 staining in primary tumor sections that otherwise (controls) showed very low and localized levels of GADD34 staining. +The proliferation of GADD34 cells was significantly increased (Figure 11B). Measurement in primary tumors served as a surrogate biomarker for the abemaciclib-induced silencing-associated UPR. As expected, treatment with LY4 abolished GADD34 expression in the primary tumors of treated animals (Figure 11B). Sequential treatment of mice with abemaciclib (dormancy induction phase) followed by LY4 (dormant DCC eradication phase) resulted in the same reduction in macrometastatic burden observed with LY4 monotherapy (Figure 11C). However, the combination also almost completely eliminated the presence of micrometastases (Figure 11D) and, as seen with the monotherapy, significantly reduced the number of quiescent, single disseminated cancer cells (Figure 11E). Collectively, these results support the rationale for sequential combination of cytostatic agents, such as CDK inhibitors, followed by LY4 as a promising therapeutic strategy to prevent metastasis by targeting quiescent DCCs that reactivate and seed these lesions.

[0172] Example 7 - Discussion of Examples 2 to 6 HER2 + Multiple studies in breast cancer models have demonstrated that HER2 +We conclude that breast cancer tumorigenesis depends on PERK signaling for survival and adaptation (Bobrovnikova-Marjon et al., “PERK Promotes Cancer Cell Proliferation and Tumor Growth by Limiting Oxidative DNA Damage,” Oncogene 29(27):3881-95 (2010); Singh et al., “HER2-mTOR Signaling-Driven Breast Cancer Cells Require ER-Associated Degradation to Survive,” Sci. Signal. 8:ra52 (2015); Avivar-Valderas et al., “PERK Integrates Autophagy and Oxidative Stress Responses to Promote Survival During Extracellular Matrix Detachment,” Mol. Cell. Biol. 31:3616-3629 (2011); and Avivar-Valderas et al., “Regulation of Autophagy During ECM Detachment is Linked to a Selective Inhibition of mTORC1 by PERK,” Oncogene 32(41):4932-40 (2013), each of which is incorporated herein by reference in its entirety. Interestingly, quiescent tumor cells present at the surgical margins and also in target organs as dormant disseminated cancer cells (Bragado et al., “TGF-Beta2 Dictates Disseminated Tumor Cell Fate in Target Organs Through TGF-Beta-RIII and P38Alpha / Beta Signaling,” Nat. Cell. Biol. 15:1351-1361 (2013); Chery et al., “Characterization of Single Disseminated Prostate Cancer Cells Reveals Tumor Cell Heterogeneity and Identifies Dormancy Associated Pathways,” Oncotarget 5(20):9939-51 (2014); Sosa et al., “Mechanisms of Disseminated Cancer Cell Dormancy: An Awakening Field,” Nat. Rev. Cancer 14:611-622 (2014); and Sosa et al., “NR2F1 Controls Tumor Cell Dormancy Via SOX9- and RARbeta-Driven Quiescence Programmes,” Nat. Commun. 6:6170 (2015), each of which is incorporated herein by reference in its entirety), have also been found to activate PERK signaling along with other ER stress pathways for survival (Adomako et al., “Identification of Markers that Functionally Define a Quiescent Multiple Myeloma Cell Sub-Population Surviving Bortezomib Treatment,” BMC Cancer 15:444 (2015); Ranganathan et al., “Dual Function of Pancreatic Endoplasmic Reticulum Kinase in Tumor Cell Growth Arrest and Survival,” Cancer Res. 68:3260-3268 (2008); Ranganathan et al., “Functional Coupling of p38-Induced Up-Regulation of BiP and Activation of RNA-Dependent Protein Kinase-Like Endoplasmic Reticulum Kinase to Drug Resistance of Dormant Carcinoma Cells,” Cancer Res. 66:1702-1711 (2006); Schewe et al., “ATF6alpha-Rheb-mTOR Signaling Promotes Survival of Dormant Tumor Cells In Vivo,” Proc. Nat'l. Acad. Sci. USA 105:10519-10524(2008); Schewe et al., “Inhibition of eIF2alpha Dephosphorylation Maximizes Bortezomib Efficiency and Eliminates Quiescent Multiple Myeloma Cells Surviving Proteasome Inhibitor Therapy,” Cancer Res. 69:1545-1552 (2009); and Chery et al., “Characterization of Single Disseminated Prostate Cancer Cells Reveals Tumor Cell Heterogeneity and Identifies Dormancy Associated Pathways,” Oncotarget 5(20):9939-51 (2014), each of which is incorporated herein by reference in its entirety. Recently, Pommier et al., "Unresolved Endoplasmic Reticulum Stress Engenders Immune-Resistant, Latent Pancreatic Cancer Metastases," Science 360(6394):eaao4908 (2018) (incorporated herein by reference in its entirety) substantiated this by showing that pancreatic DCCs in the liver (and other models) activate the UPR during quiescence. This level of reproducibility across a variety of cancers and models suggests a high degree of biological plausibility for this biology.

[0173] The Examples herein demonstrate that the PERK inhibitor LY4 can selectively target HER2 dependency in DCCs and primary lesions. A particularly noteworthy finding is the metastasis-inhibitory effect of LY4. In the MMTV-HER2 model, as in patients, metastasis can be asynchronous with the primary tumor, sometimes occurring alongside occult primary lesions, and some metastasis may even begin before overt tumor detection (Husemann et al., "Systemic Spread Is an Early Step in Breast Cancer," Cancer Cell 13:58-68 (2008); Pavlidis et al., "Cancer of Unknown Primary (CUP)," Crit. Rev. Oncol. Hematol. 54:243-250 (2005); Harper et al., "Mechanism of Early Dissemination and Metastasis in Her2 +Mammary Cancer,” Nature 540:588-592 (2016); and Hosseini et al., “Early Dissemination Seeds Metastasis in Breast Cancer,” Nature 540:552-558 (2016), incorporated herein by reference in its entirety. LY4 treatment reduced all metastases, both those with early onset (before overt tumors were evident) and those occurring simultaneously with overt primary tumor growth (Figure 8). This is important because it demonstrates that the effect on metastasis does not solely depend on LY4 reducing primary tumor burden. Surprisingly, LY4 treatment reduced metastatic burden by eliminating nonproliferating, solitary or small clusters of P-Rb-negative DTCs. Imaging and single-cell multiplex qPCR revealed that these DCCs more frequently upregulated GADD34 (protein) and a larger set of ER stress genes, including PERK itself, and also displayed a quiescent phenotype manifested by the upregulation of several negative regulators of cell proliferation. It should be noted that part of the PERK-induced ER stress program involves transcriptional regulation, while another part is preferential translation of genes containing upstream ORFs, such as ATF4 and GADD34 (Young et al., "Upstream Open Reading Frames Differentially Regulate Gene Specific Translation in the Integrated Stress Response,” J. Biol. Chem. 291:16927-16935 (2016), incorporated herein by reference in its entirety).Similarly, inhibition of cyclin D1 translation has been shown to result in UPR-induced G1 arrest (Brewer et al., "Mammalian Unfolded Protein Response Inhibits Cyclin D1 Translation and Cell-Cycle Progression," Proc. Natl. Acad. Sci. 96:8505-8510 (1999), incorporated herein by reference in its entirety). Human HER2. + 3D organogenesis experiments using cancer cell lines confirmed the selective killing of quiescent single cancer cells by LY4. These data suggest that quiescent DCCs likely depend on PERK signaling for survival. Similarly, a subpopulation of human metastatic cells derived from breast cancer patients also showed a negative correlation between GADD34 and Ki67, confirming the relationship found in mice. These data, along with NR2F1 (Borgen et al., "NR2F1 Stratifies Dormant Disseminated Tumor Cells in Breast Cancer Patients," Breast Cancer Research 20:120 (2018)), incorporated herein by reference in its entirety), suggest that GADD34 alone or in combination with NR2F1 may provide a robust biomarker set for dormant / UPRhigh DCCs and thus may be an indicator for selecting patients for treatment.

[0174] The discovery of targets and drugs that can eradicate dormant DCCs would be highly significant. Dormant DCCs are known to evade antiproliferative therapies through active and passive mechanisms (Aguirre-Ghiso et al., “Metastasis Awakening: Targeting Dormant Cancer,” Nat. Med. 19:276-277 (2013); Naumov et al., “Ineffectiveness of Doxorubicin Treatment on Solitary Dormant Mammary Carcinoma Cells or Late-Developing Metastases,” Breast Cancer Res. Treat. 82(3):199-206 (2003); Oshimori et al., “TGF-Beta Promotes Heterogeneity and Drug Resistance in Squamous Cell Carcinoma,” Cell 160:963-976 (2015); and Fluegen et al., “Phenotypic Heterogeneity of Disseminated Tumor Cells is Preset by Primary Tumor Hypoxic Microenvironments,” Nat. Cell Biol. 19(2):120-132 (2017), each of which is incorporated herein by reference in its entirety.Eradication of DCCs in the bone marrow, which are also generally dormant (Bragado et al., “TGF-Beta2 Dictates Disseminated Tumor Cell Fate in Target Organs Through TGF-Beta-RIII and P38Alpha / Beta Signaling,” Nat. Cell. Biol. 15:1351-1361 (2013); Chery et al., “Characterization of Single Disseminated Prostate Cancer Cells Reveals Tumor Cell Heterogeneity and Identifies Dormancy Associated Pathways,” Oncotarget 5(20):9939-51 (2014); Ghajar et al., “The Perivascular Niche Regulates Breast Tumor Dormancy,” Nat. Cell Biol. 15:807-817 (2013); and Husemann et al., “Systemic Spread Is an Early Step in Breast Cancer,” Cancer Cell 13:58-68 (2008), incorporated herein by reference in its entirety), further strengthens the concept of PERK inhibition as an anti-dormant DCC therapy (Aguirre-Ghiso et al., "Metastasis Awakening: Targeting Dormant Cancer," Nat. Med. 19:276-277 (2013), incorporated herein by reference in its entirety), which may be used in the adjuvant setting to eliminate dormant minimal residual disease (Aguirre-Ghiso et al., "Metastasis Awakening: Targeting Dormant Cancer," Nat. Med. 19:276-277 (2013), incorporated herein by reference in its entirety).

[0175] The exact mechanism by which PERK kinase inhibition interferes with tumor growth is unclear. One possible mechanism is impaired adaptation to stress imposed by proteotoxicity (Singh et al., "HER2-mTOR Signaling-Driven Breast Cancer Cells Require ER-Associated Degradation to Survive," Sci. Signal. 8:ra52 (2015), incorporated herein by reference in its entirety). The results described herein demonstrate that LY4 reduced phospho-HER2 levels in vivo and reduced the number of active receptors in the membrane by enhancing endocytosis. No changes in HER2 proteolysis were observed. Regarding how PERK precisely regulates HER2 membrane localization or endocytosis, it is possible that receptor internalization allows for better or faster receptor dephosphorylation or reduces the opportunity for receptor activation, thereby reducing downstream signaling. Receptor endocytosis has been shown to reduce the signaling output of many receptors localized to the plasma membrane by physically reducing the concentration of cell surface receptors (Sorkin and Zastrow, "Endocytosis and Signaling: Intertwining Molecular Networks," Nat. Rev. Mol. Cell Biol. 10:609-22 (2009), incorporated herein by reference in its entirety).

[0176] The results herein further demonstrate that LY4 induced a differentiated phenotype in early lesions. However, in established tumors, LY4 as a single agent drove tumors into stasis or regression. This suggests that early on, HER2 + We show that deregulation of PERK signaling in early lesions is more likely to be associated with loss of the differentiation program through undetermined mechanisms, and that even as tumor biology changes to become hyperproliferative, the dependency on PERK persists in these HER2-positive tumors. +It is still highly dependent in tumors, which may be related to how HER2 function changes during progression, initially deregulating primarily morphogenetic programs leading to anoikis resistance and dissemination, but later engaging primarily proliferative and survival programs.

[0177] The results described herein also point to the value of combining standard antiproliferative therapy with LY4 to eliminate remaining quiescent cells. Such an approach was tested using the CDK4 / 6 inhibitor abemaciclib followed by LY4, revealing improved anti-metastatic efficacy. Encouragingly, the LY4 dose used did not significantly affect glucose levels, bone marrow or peripheral blood cell counts, or feeding behavior in tumor- or cancer-free mice. This indicates that the dose used robustly disrupted tumor growth and metastasis by eradicating dormant DCCs, while not affecting normal host organ function. Dormancy / UPR high Because DCCs have also been shown to downregulate MHC-I surface expression (Pommier et al., "Unresolved Endoplasmic Reticulum Stress Engenders Immune-Resistant, Latent Pancreatic Cancer Metastases," Science 360(6394):eaao4908 (2018) (incorporated herein by reference in its entirety), LY4 may also assist the adaptive immune response in targeting DCCs, and possibly pre-existing tumors as well. This is the possibility addressed in the present study. The results described herein open the door to the use of anti-dormant DCC survival therapies as a new way to target metastatic disease. This would enable targeting the full phenotypic diversity of disseminated disease, which may include proliferative, slow-cycling, and dormant DCCs (Aguirre-Ghiso et al., "Metastasis Awakening: Targeting Dormant Cancer," Nat. Med. 19:276-277). (2013), which is incorporated herein by reference in its entirety).

[0178] Example 8 – Combination of the CDK4 / 6 inhibitor Abemaciclib with the PERK inhibitor LY4 in melanoma cell lines Because CDK4 / 6 inhibitors have been shown to induce cell cycle arrest, and LY4 induces cell death in dormant, cell cycle-arrested DTCs (Figure 12A), we next investigated whether the combination of CDK4 / 6 inhibitors and LY4 reduces cell viability in in vitro cell lines. The CDK4 / 6 inhibitor abemaciclib has been shown to inhibit the growth of WM35 melanoma cells in both 2D and 3D in vitro cell cultures. Pretreatment with 50 nM abemaciclib for 1 week followed by short-term in vitro treatment (48 h) with 2 μM LY4 (2D) reduced the viability of Braf-mutant melanoma WM35 cells compared to cells treated with 2 μM LY4 alone (Figure 12B). In in vitro 3D cultures, the addition of 2 μM LY4 following 1 week of abemaciclib pretreatment had a further effect on reducing cell viability (Figure 12C). These results, shown in Figure 12C, suggest that abemaciclib pretreatment can induce growth arrest and partial cell death in 3D cell cultures. If so, the addition of LY4 may enhance the cell death effect. This is consistent with the notion that abemaciclib-arrested cells can upregulate the ER stress response, as evidenced by upregulation of GADD34 (Figure 12G), and become sensitive to LY4.

[0179] In melanoma cells, the abemaciclib-resistant phenotype emerges after 4–5 weeks of continuous treatment. Co-treatment of cells with LY4 and abemaciclib reduces the number of viable abemaciclib-resistant cells in 2D cell culture, but not as strongly as would be expected from the experimental implementation of these 2D cultures. However, in abemaciclib-resistant cells, LY4 has a further effect of reducing viability in 3D cell culture after continuous treatment with abemaciclib (Figures 12D–12E). These data indicate that melanoma CDK4 / 6 inhibitor-resistant cells still rely on the PERK-mediated ER stress response for survival. In in vitro 3D cultures, the addition of LY4 after 5 weeks of abemaciclib pretreatment has a further effect of reducing cell viability (Figure 12F), as measured by apoptosis of DAPI-uptake cells. In 2D culture, resistant cells proliferated similarly to control cells, but in 3D culture, abemaciclib pretreatment appeared to induce cell death (Figure 12F).

[0180] Example 9 - BMP7-F9 induces and maintains dormancy in DTC (HNSCC) BMP7-F9 reduces the ERK / p38 activity ratio and induces various mRNAs of the dormancy signature (Figures 13A-13C). Figure 13A shows that treatment with 2 ng / ml, 5 ng / ml, and 10 ng / ml BMP7-F9 (second, third, and fourth gray bars, respectively; control, first black bar) reduced the ERK / p38 activity ratio compared to the control, as determined by Western blot in HEp3 HNSCC cells. The effect on the ERK / p38 activity ratio was observed after 2, 6, and 24 hours (second to fourth vertical groups). ERK activity was stimulated by BMP7 in the first 30 minutes (first vertical group). Figure 13B shows that BMP7-F9 treatment (10 ng / ml BMP7-F9, 24 hours) induces DEC2, p53, and p27 mRNAs, which encode dormancy signature genes. Figure 13C shows that BMP7-F9 treatment of the same cells induces nuclear accumulation of the potent dormancy-inducing transcription factor NR2F1, as determined by immunofluorescence (10 ng / ml, 24 hours). The difference between Figure 13A and Figure 13B is p<0.05, calculated by Student's t-test.

[0181] BMP7-F9 induces growth arrest in T-HEp3 cells in vitro and in vivo (Figures 14A-14E). Figure 14A shows that BMP7-F9 treatment of T-HEp3 cells inhibited proliferation for 48 hours in vitro, as determined by cell titer blue assay (RFU, relative fluorescence units). Figure 14B is a schematic diagram of the in vivo experimental procedure used in Figures 14C-14D. T-HEp3 cells were pretreated with BMP7-F9 for 24 hours in vitro and then inoculated into chick embryo chorioallantoic membranes (CAMs) (Figure 14C). After daily treatment with vehicle or BMP7-F9 (50 ng / ml) in vivo, tumors were harvested, and the number of HEp3 HNSCC cells / tumor was quantified (Figure 14D), as were the levels of P-H3 (Figure 14E).

[0182] NSG mice were treated for 3 and 6 weeks according to the protocol in Figure 15A. At these time points, the percentage of local recurrence and DTC appearance were scored. The results table corresponding to Figure 15B shows that BMP7 limits the local recurrence rate (Table 9) and the appearance of DTCs in the lungs (Table 10) after tumor surgery, as shown below (Table 11).

[0183] Table 9. Effect of BMP7-F9 in the adjuvant setting on local recurrence at the surgical margin. TIFF0007719135000020.tif48155

[0184] Table 10. Effect of BMP7-F9 on the appearance of DCCs in the lungs TIFF0007719135000021.tif48155

[0185] Table 11: GFP in the same experiment shown in Figures 15A and 15B and Tables 9 and 10 + / Vimentin + Median tumor cells / lung TIFF0007719135000022.tif55156

[0186] HEp3-GFP HNSCC tumors were grown to approximately 300 mm 3 The tumors were allowed to grow until they reached approximately 600 mm in the neoadjuvant setting with 50 μg / kg BMP7-F9. 3The tumors were then surgically removed. One to two days after surgery, adjuvant treatment with BMP7-F9 was continued for an additional four weeks. The animals were then euthanized, and the DCC burden in the lungs was scored using a fluorescent microscope. We observed that BMP7 limited the occurrence of local and distant recurrence after tumor surgery. NSG mice were treated according to the protocol in Figure 15A for four weeks. At those time points, the percentage of local recurrence and DCC appearance was scored. After treatment, the number of GFP-positive cells in the excised lungs was scored. This represents the magnitude of the DCC burden in the lungs, which was significantly reduced by BMP7-F9 treatment. Note the median DCC burden was one log lower, and BMP-7 clearly cured three out of seven animals of DCC.

[0187] The effects of BMP7-F9 in the neoadjuvant and adjuvant setting on local recurrence at the surgical margin (Table 12) and on the appearance of DCC in the lung (Table 13) are shown below. The results are tabulated from Figure 15C, where mice were treated as shown in Figure 15A, but with 4 weeks of adjuvant treatment. After treatment, the number of GFP-positive cells in the excised lungs was scored. The results show that BMP7, with neoadjuvant and adjuvant treatment, limits the local recurrence rate after tumor surgery (Table 12) and the appearance of DCC in the lung (Table 13). Table 14 shows the magnitude of the DCC burden in the lung, which is significantly reduced by BMP7-F9 treatment. Note that the median DCC burden is 1 log lower and that BMP-7 clearly cured 3 of 7 animals of DCC.

[0188] Table 12. Effect of BMP7-F9 in the neoadjuvant + adjuvant setting on local recurrence at the surgical margin. TIFF0007719135000023.tif39155

[0189] (Table 13) Appearance of intrapulmonary DCC TIFF0007719135000024.tif39155

[0190] Table 14: Mouse GFP reported in Tables 12 and 13 + / Vimentin + Median tumor cells / lung TIFF0007719135000025.tif46156

[0191] While preferred embodiments have been shown and described in detail herein, it will be apparent to those skilled in the art that various modifications, additions, substitutions, etc. may be made therein without departing from the spirit of the invention and are therefore deemed to be within the scope of the invention as defined in the following claims.

[0192] Sequence information SEQUENCE LISTING <110> ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI <120> METHODS OF TREATING MINIMAL RESIDUAL CANCER <150> US 62 / 648,166 <151> 2018-03-26 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 431 <212> PRT <213> Homo sapiens <400> 1 Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 5 10 15 Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 20 25 30 Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser 35 40 45 Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu 50 55 60 Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro 65 70 75 80 Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly 85 90 95 Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser 100 105 110 Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr 115 120 125 Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys 130 135 140 Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu 145 150 155 160 Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile 165 170 175 Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile 180 185 190 Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu 195 200 205 Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu 210 215 220 Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg 225 230 235 240 His Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser 245 250 255 Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn 260 265 270 Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe 275 280 285 Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser 290 295 300 Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu 305 310 315 320 Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr 325 330 335 Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu 340 345 350 Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn 355 360 365 Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His 370 375 380 Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln 385 390 395 400 Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile 405 410 415 Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425 430 <210> 2 <211> 139 <212> PRT <213> Homo sapiens <400> 2 Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 <210> 3 <211> 139 <212> PRT <213> Homo sapiens <220> <221> misc_feature <222> (33)..(33) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (37)..(37) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (60)..(60) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (65)..(65) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (86)..(87) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (89)..(89) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (91)..(91) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (93)..(94) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (110)..(110) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (114)..(114) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (120)..(120) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (128)..(128) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (132)..(132) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (134)..(134) <223> Xaa can be any naturally occurring amino acid <400> 3 Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 30 Xaa Gln Arg Gln Xaa Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Xaa Gly Tyr Ala Ala 50 55 60 Xaa Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Only Thr Asn His No More Gln Only Leu Only His Only Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Ala Pro Thr Gln Leu Xaa Ala Ile 100 105 110 Your Tyr Phe Asp Asp Your Ser Asn Val Ileu Lys Lys 115 120 125 Arg Asn Met Stone Val Stone Ala Cys Gly Cys His 130 135 <210> 4 <211> 139 <212> PRT <213> Homo sapiens <400> 4 Serving Thr Gly Serving Lys Gln Arg Serving Gln Asn Arg Serving Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Val Ile Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Gly Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 <210> 5 <211> 139 <212> PRT <213> Homo sapiens <400> 5 Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 Gly Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Leu Val Gln Ala Leu Val His Phe Ile Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Gly Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 <210> 6 <211> 139 <212> PRT <213> Homo sapiens <400> 6 Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 Gly Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Leu Val Gln Thr Leu Val His Phe Ile Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Gly Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Phe 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 <210> 7 <211> 139 <212> PRT <213> Homo sapiens <400> 7 Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 Gly Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Leu Val Gln Thr Leu Val His Phe Ile Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Gly Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Trp 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 <210> 8 <211> 139 <212> PRT <213> Homo sapiens <400> 8 Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 Gly Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Leu Val Gln Thr Leu Val His Val Ile Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Gly Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Trp 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 <210> 9 <211> 139 <212> PRT <213> Homo sapiens <400> 9 Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 Gly Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Ile Val Gln Ala Leu Val His Phe Ile Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Gly Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Phe 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 <210> 10 <211> 139 <212> PRT <213> Homo sapiens <400> 10 Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 Gly Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Leu Val Gln Thr Leu Val His Phe Ile Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Gly Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 <210> 11 <211> 139 <212> PRT <213> Homo sapiens <400> 11 Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 Gly Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile 100 105 110 Ser Met Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 <210> 12 <211> 402 <212> PRT <213> Homo sapiens <400> 12 Asp Phe Ser Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg 1 5 10 15 Leu Arg Ser Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile 20 25 30 Leu Gly Leu Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn 35 40 45 Ser Ala Pro Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu 50 55 60 Glu Gly Gly Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala 65 70 75 80 Val Phe Ser Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His 85 90 95 Phe Leu Thr Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu 100 105 110 His Asp Lys Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg 115 120 125 Phe Asp Leu Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu 130 135 140 Phe Arg Ile Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr 145 150 155 160 Phe Arg Ile Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu 165 170 175 Ser Asp Leu Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu 180 185 190 Gly Trp Leu Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val 195 200 205 Asn Pro Arg His Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp 210 215 220 Gly Gln Ser Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly 225 230 235 240 Pro Gln Asn Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu 245 250 255 Val His Phe Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln 260 265 270 Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn 275 280 285 Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His 290 295 300 Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile 305 310 315 320 Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe 325 330 335 Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr 340 345 350 Leu Val His Val Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala 355 360 365 Pro Thr Gln Leu Gly Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser 370 375 380 Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly 385 390 395 400 Cys His <210> 13 <211> 402 <212> PRT <213> Homo sapiens <400> 13 Asp Phe Ser Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg 1 5 10 15 Leu Arg Ser Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile 20 25 30 Leu Gly Leu Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn 35 40 45 Ser Ala Pro Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu 50 55 60 Glu Gly Gly Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala 65 70 75 80 Val Phe Ser Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His 85 90 95 Phe Leu Thr Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu 100 105 110 His Asp Lys Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg 115 120 125 Phe Asp Leu Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu 130 135 140 Phe Arg Ile Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr 145 150 155 160 Phe Arg Ile Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu 165 170 175 Ser Asp Leu Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu 180 185 190 Gly Trp Leu Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val 195 200 205 Asn Pro Arg His Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp 210 215 220 Gly Gln Ser Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly 225 230 235 240 Pro Gln Asn Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu 245 250 255 Val His Phe Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln 260 265 270 Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn 275 280 285 Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His 290 295 300 Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile 305 310 315 320 Ala Pro Glu Gly Tyr Ala Ala Gly Tyr Cys Glu Gly Glu Cys Ala Phe 325 330 335 Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Leu Val Gln Ala 340 345 350 Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala 355 360 365 Pro Thr Gln Leu Gly Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser 370 375 380 Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly 385 390 395 400 Cys His <210> 14 <211> 402 <212> PRT <213> Homo sapiens <400> 14 Asp Phe Ser Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg 1 5 10 15 Leu Arg Ser Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile 20 25 30 Leu Gly Leu Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn 35 40 45 Ser Ala Pro Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu 50 55 60 Glu Gly Gly Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala 65 70 75 80 Val Phe Ser Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His 85 90 95 Phe Leu Thr Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu 100 105 110 His Asp Lys Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg 115 120 125 Phe Asp Leu Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu 130 135 140 Phe Arg Ile Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr 145 150 155 160 Phe Arg Ile Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu 165 170 175 Ser Asp Leu Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu 180 185 190 Gly Trp Leu Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val 195 200 205 Asn Pro Arg His Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp 210 215 220 Gly Gln Ser Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly 225 230 235 240 Pro Gln Asn Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu 245 250 255 Val His Phe Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln 260 265 270 Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn 275 280 285 Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His 290 295 300 Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile 305 310 315 320 Ala Pro Glu Gly Tyr Ala Ala Gly Tyr Cys Glu Gly Glu Cys Ala Phe 325 330 335 Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Leu Val Gln Thr 340 345 350 Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala 355 360 365 Pro Thr Gln Leu Gly Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser 370 375 380 Asn Val Ile Leu Lys Lys Phe Arg Asn Met Val Val Arg Ala Cys Gly 385 390 395 400 Cys His <210> 15 <211> 402 <212> PRT <213> Homo sapiens <400> 15 Asp Phe Ser Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg 1 5 10 15 Leu Arg Ser Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile 20 25 30 Leu Gly Leu Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn 35 40 45 Ser Ala Pro Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu 50 55 60 Glu Gly Gly Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala 65 70 75 80 Val Phe Ser Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His 85 90 95 Phe Leu Thr Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu 100 105 110 His Asp Lys Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg 115 120 125 Phe Asp Leu Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu 130 135 140 Phe Arg Ile Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr 145 150 155 160 Phe Arg Ile Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu 165 170 175 Ser Asp Leu Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu 180 185 190 Gly Trp Leu Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val 195 200 205 Asn Pro Arg His Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp 210 215 220 Gly Gln Ser Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly 225 230 235 240 Pro Gln Asn Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu 245 250 255 Val His Phe Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln 260 265 270 Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn 275 280 285 Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His 290 295 300 Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile 305 310 315 320 Ala Pro Glu Gly Tyr Ala Ala Gly Tyr Cys Glu Gly Glu Cys Ala Phe 325 330 335 Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Leu Val Gln Thr 340 345 350 Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala 355 360 365 Pro Thr Gln Leu Gly Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser 370 375 380 Asn Val Ile Leu Lys Lys Trp Arg Asn Met Val Val Arg Ala Cys Gly 385 390 395 400 Cys His <210> 16 <211> 402 <212> PRT <213> Homo sapiens <400> 16 Asp Phe Ser Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg 1 5 10 15 Leu Arg Ser Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile 20 25 30 Leu Gly Leu Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn 35 40 45 Ser Ala Pro Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu 50 55 60 Glu Gly Gly Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala 65 70 75 80 Val Phe Ser Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His 85 90 95 Phe Leu Thr Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu 100 105 110 His Asp Lys Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg 115 120 125 Phe Asp Leu Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu 130 135 140 Phe Arg Ile Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr 145 150 155 160 Phe Arg Ile Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu 165 170 175 Ser Asp Leu Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu 180 185 190 Gly Trp Leu Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val 195 200 205 Asn Pro Arg His Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp 210 215 220 Gly Gln Ser Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly 225 230 235 240 Pro Gln Asn Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu 245 250 255 Val His Phe Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln 260 265 270 Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn 275 280 285 Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His 290 295 300 Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile 305 310 315 320 Ala Pro Glu Gly Tyr Ala Ala Gly Tyr Cys Glu Gly Glu Cys Ala Phe 325 330 335 Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Leu Val Gln Thr 340 345 350 Leu Val His Val Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala 355 360 365 Pro Thr Gln Leu Gly Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser 370 375 380 Asn Val Ile Leu Lys Lys Trp Arg Asn Met Val Val Arg Ala Cys Gly 385 390 395 400 Cys His

Claims

1. A pharmaceutical composition for treating minimal residual cancer in a subject, comprising a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor, The protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor has the chemical structure 2-amino-5-[4-[[(2R)-2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N-isopropyl-pyridine-3-carboxamide having the formula disseminated cancer cells (DCCs) in a subject are contacted with the protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor, thereby eradicating DCCs in the subject and treating minimal residual cancer in the subject. The pharmaceutical composition.

2. 10. The pharmaceutical composition of claim 1, wherein the subject has been diagnosed with breast cancer, multiple myeloma, lung cancer, non-small cell lung cancer, brain cancer, cervical cancer, mantle cell lymphoma, leukemia, hepatocellular carcinoma, prostate cancer, melanoma, skin cancer, head and neck cancer, thyroid cancer, glioblastoma, neuroblastoma, or colorectal cancer.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the cancer is a breast cancer selected from invasive breast cancer, ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and inflammatory breast cancer.

4. The breast cancer is HER2 + The pharmaceutical composition of claim 3, wherein the cancer is breast cancer.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the subject has been diagnosed with disseminated cancer cells and / or non-metastatic cancer.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, for use in combination with a chemotherapeutic agent, an immunotherapeutic agent, an epigenetic agent, or ionizing radiation.

7. 7. The pharmaceutical composition of claim 6, wherein a chemotherapeutic agent is used and the chemotherapeutic agent is an anti-HER2 chemotherapeutic agent selected from trastuzumab (Herceptin®) and lapatinib (Tykerb®).

8. 7. The pharmaceutical composition of claim 6, wherein a chemotherapeutic agent is used and the chemotherapeutic agent is selected from anthracyclines, taxanes, kinase inhibitors, antibodies, fluoropyrimidines, and platinum drugs.

9. 7. The pharmaceutical composition of claim 6, wherein an immunotherapeutic agent is used and the immunotherapeutic agent is selected from an immune checkpoint inhibitor, an interferon, or a tumor vaccine.

10. 7. The pharmaceutical composition of claim 6, wherein an epigenetic agent is used and the epigenetic agent is selected from a histone deacetylase (HDAC) inhibitor, 5-azacytidine, retinoic acid, arsenic trioxide, a Zeste2 enhancer polycomb repressive complex 2 subunit (EZH2) inhibitor, and a bromodomain ("BRD") inhibitor.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein administration of the PERK inhibitor to a subject results in contact of disseminated cancer cells (DCCs) within the subject with the PERK inhibitor.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the subject has been diagnosed with DCC.

13. The DCC is NR2F1 + 13. The pharmaceutical composition of claim 12, wherein

14. 14. The pharmaceutical composition of claim 12 or claim 13, wherein the DCC is phospho-PERK active.

15. The DCCs were bone morphogenetic protein receptor-positive (BMPR + 15. The pharmaceutical composition according to any one of claims 12 to 14, wherein

16. The pharmaceutical composition of claim 15, used in conjunction with a bone morphogenetic protein 7 (BMP7) derivative protein, wherein DCCs in the subject are contacted with the bone morphogenetic protein 7 (BMP7) derivative protein.

17. The pharmaceutical composition of claim 16, wherein administration of the BMP7 derivative protein to a subject results in contact of disseminated cancer cells (DCCs) within the subject with the BMP7 derivative protein.

18. 18. The pharmaceutical composition of claim 16 or 17, wherein the BMP7 derivative protein is BMP7-F9.

19. 19. The pharmaceutical composition of any one of claims 1 to 18, wherein the PERK inhibitor does not inhibit EIF2AK1, EIF2AK2 or EIF2AK4.

20. The pharmaceutical composition of any one of claims 1 to 19, wherein the subject is a human.

21. The pharmaceutical composition of any one of claims 1 to 20, wherein the subject is in cancer remission.

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