Method for treating minimal residual cancer

Abstract

The present application discloses methods for treating minimal residual cancer in a subject. The methods involve contacting disseminated cancer cells (DCCs) in the subject with a bone morphogenetic protein 7 (BMP7)-derived protein, wherein the contacting induces or maintains dormancy in the contacted DCCs in the subject to treat minimal residual cancer in the subject. The present application also discloses methods involving contacting DCCs in a subject with a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor selected from LY2, LY3, and LY4, wherein the contacting eradicates the DCCs in the subject to treat minimal residual cancer in the subject.

Description

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 648,166, filed Mar. 26, 2018, the entire content of which is hereby incorporated by reference.

[0002] This invention was made with government support under R01 CA109182, U54CA16131, and P30 CA196521 awarded by the National Institutes of Health / National Cancer Institute, and BC 132674 awarded by the Department of Defense Congressionally Directed Medical Research Programs. The government has certain rights in the invention. Field of the Invention

[0003] The present invention relates to methods of treating minimal residual cancer in a subject. Background Art

[0004] Protein unfolding in the endoplasmic reticulum (“ER”) lumen activates three major pathways, PERK, IRE1α, and ATF6, also known as the unfolded protein response (“UPR”), which enables cells to correct and survive this stress (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 is a mechanism that enables tumor cells to respond to increased translational loads caused by signals such as oncogenes and hypoxia, and to the demands of the endoplasmic reticulum and oxidative conditions (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); and Sequeira et al., “Inhibition of eIF2alpha Dephosphorylation Inhibits ErbB2-Induced Deregulation of Mammary Acinar Morphogenesis,” BMC Cell Biol. 10:64(2009)). The oncogene-activated pathway enhances the loading of ER client proteins by activating mTOR signaling and translation initiation (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)). It has further been shown that the PERK and IRE1α-XBP-1 pathways 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., “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. U.S.A. 105:10519-10524(2008); and Ye et al., “The GCN2-ATF4 Pathway is Critical for Tumour Cell Survival and Proliferation in Response to Nutrient Deprivation,” EMBO J. 29:2082-2096(2010)), indicating that the UPR allows adaptation to changing environments.

[0005] Activation of PERK coordinates antioxidant and autophagy responses to protect mammary epithelial cells during loss of adhesion 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 ATF4 and CHOP transcriptional programs associated with rapid activation of the LKB1-AMPK-TSC2 pathway that 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)) (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)).Human DCIS lesions exhibit 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 the mammary epithelium delays breast carcinogenesis induced by the HER2 proto-oncogene (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. U.S.A. 105:16314-16319 (2008)). In addition, HER2 can elevate the proteotoxicity level in tumor cells, activate JNK and IRE signaling and phosphorylate HER2. +Cancer cells are able to cope with this stress (Singh et al., “HER2-mTOR Signaling-Driven Breast Cancer Cells Require ER-Associated Degradation to Survive,” Sci. Signal. 8:ra52 (2015)). Thus, 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 human breast tumors with HER2 amplification exhibit upregulation of PERK mRNA, which further supports the view that HER2 + tumors may rely on PERK and / or other UPR pathways for survival.

[0006] Quiescent tumor cells have also been shown to rely 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. U.S.A. 105:10519 - 10524 (2008)). Quiescent pancreatic disseminated cancer cells (“DCCs”) in the liver also exhibit a PERK - dependent UPR, which is associated with loss of E - cadherin expression and down - regulation of MHC - 1, facilitating immune evasion during dormancy (Pommier et al., “Unresolved Endoplasmic Reticulum Stress Engenders Immune - Resistant, Latent Pancreatic Cancer Metastases,” Science 360(6394):eaao4908 (2018), the entire content of which is incorporated herein by reference). 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 + Mammary Cancer,” Nature 540:588 - 592 (2016)), but the association with the UPR was not tested. Together, these data suggest that the UPR can serve as a stress and immune microenvironment adaptation survival mechanism for DCCs.

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

[0009] One aspect of the present disclosure relates to a method of treating minimal residual cancer in a subject. The method involves contacting disseminated cancer cells (DCCs) in the subject with a bone morphogenetic protein 7 (“BMP7”) - derived protein, wherein the contacting induces or maintains dormancy in the contacted DCCs in the subject to treat minimal residual cancer in the subject. The methods of this aspect can be used to prevent minimal residual cancer from developing into an invasive growth in the subject.

[0010] Another aspect relates to a method of treating minimal residual cancer in a subject, the method involving 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, wherein the contacting eradicates the DCCs in the subject to treat minimal residual cancer in the subject.

[0011] Yet another aspect relates to a method of treating advanced cancer in a subject. The method involves 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, wherein the contacting eradicates the DCCs in the subject to treat advanced cancer in the subject.

[0012] Hereinafter, it was demonstrated that LY4, as a selective and potent inhibitor of PERK, can block HER2 - driven metastasis, which benefits from its ability to specifically cause the elimination of dormant DCCs. As described in the present disclosure, PERK inhibitors represent a new strategy for targeting single dormant cells during the minimal residual disease stage, which are administered alone or in combination with anti - proliferative therapies to help prevent lethal metastasis. The following description also demonstrates that bone morphogenetic - derived proteins can induce dormancy in disseminated tumor cells. Brief Description of the Drawings

[0014] Figures 1A - 1C Quiescent disseminated HER2 + cells exhibit high levels of ER stress pathway activation. Figure 1A Images of MMTV - HER2 animal lung sections stained for HER2, Ki67 (proliferation), and GADD34 (ER stress) are shown. Figure 1A The figure in [ ] shows the quantification of cells / metastases positive for both markers, shown as a percentage of total cells. Figure 1BImages of human breast cancer metastases from different locations (lymph node, liver, lung) stained for cytokeratin, Ki67 (proliferation), and GADD34 (ER stress) are shown. Figure 1B The figure in Figure 1B shows the quantification of cells / metastases positive for both markers, shown as a percentage of total cells. Figure 1C Hierarchical clustering of a high-throughput targeted gene expression (columns) map of single cells (lung disseminated tumor cells (“DTC”)) (rows) is shown.

[0015] Figures 2A - 2G It is demonstrated that the inhibition of PERK is upregulated in HER2 + cancer patients. Figure 2A The figure is a flow chart of the steps followed for single cell gene expression analysis performed with C1 and Biomark HD Fluidigm. A total of 255 DCCs and 90 primary tumor (“PT”) cells were analyzed. Figure 2B A list of genes analyzed by high-throughput qPCR is shown. Figure 2C The figure is an immunoblot showing the inhibition of PERK phosphorylation by LY series inhibitors (LY2, LY3, and LY4) and GSK2656157 (2 μM) in MCF10A-HER2 cells, which were stressed by being placed in suspension for 24 hours. * indicates non-specific bands. Figure 2D The figure shows the dose-response cell viability curve (Cell Titer Blue, CTB) of LY4 in MCF10A-HER2 cells after 48 hours in the absence (-) or presence of stress (low dose thapsigargin, Tg 2 nM). The dashed line indicates IC 50 (≈9 nM). Figure 2E The figure shows the kinase selectivity of PERK inhibitors LY4, LY2, LY3, and GSK2656157 evaluated by an enzymatic biochemical assay. Figure 2F The figure shows the effect of LY4 on total myeloid cells (in both lower limbs) in MMTV-HER2 females treated for 2 weeks. Figure 2G The figure shows the effect of LY4 on total white blood cells in MMTV-HER2 females treated for 2 weeks.

[0016] Figures 3A - 3G The figure shows that at the single disseminated tumor cell level, LY4 PERK inhibition reduces metastatic disease in the lung and bone marrow. Figure 3A The figure is an immunoblot showing the inhibition of PERK phosphorylation (T980) by PERK inhibitor LY4 (2 μM) in MCF10A-HER2 cells starved of serum overnight and treated with EGF (100 ng / ml) for 15 minutes. InFigure 3B Female (24-week-old) were injected with vehicle or LY4 (50 mpk) daily for 2 weeks. Immunohistochemistry (“IHC”) of pancreatic and mammary gland sections with antibodies against P-PERK and P-EIF2α is shown. The insets show higher magnification. Scale bar, 100 μm. + Images and quantification results (right panel) of macro-metastases (>100 cells) detected by H&E staining and quantified in 5 lung sections / animal (n = 16) are shown. Scale bar, 100 μm. p was determined by Mann-Whitney test. Figure 3C Images and quantification results (right panel) of micro-metastases (2 - 100 cells) detected by IHC staining with anti-HER2 antibody and quantified per lung section / animal ± s.d (n = 6) are shown. Scale bar, 25 μm. p was determined by Mann-Whitney test. Figure 3D Images and quantification results (right panel) of isolated disseminated tumor cells (DTCs) detected by HER2 IHC staining, classified as P-Rb Figure 3E or P-Rb + and quantified per lung section ± s.d (n = 6) are shown. Scale bar, 25 μm. p was determined by Mann-Whitney test. Arrows and circles in the images indicate an isolated DTC. - Images and quantification results (right panel) of disseminated tumor cells in the bone marrow are shown, which were detected by IF staining of CK8 / 18 and HER2 in the cell spindles of bone marrow tissues depleted of mature hematopoietic cells (n = 8). Scale bar, 25 μm. p was determined by Mann-Whitney test. Arrows indicate Her2 Figure 3F cells. + Figure 3G Representative images of ZR75.1 HER2 + cells seeded at low density (single cells) on Matrigel, which were engineered to express Dendra-tagged H2B protein. At day 0, the Dendra tag (green fluorescence) was photo-converted to red fluorescence with a single UV light pulse and used as a quiescence indicator. From day 2 to day 8, the wells were treated with vehicle (DMSO) or LY4 (2 μM). The graphs show the percentage of live cells measured as ± s.d at day 8 (n = 4). p was determined by Student's t-test.

[0017] Figures 4A - 4F The effect of LY4 treatment on metastases and circulating tumor cells (“CTCs”) is shown. Figure 4A ​Shows the benchmarked area of single macrometastases in vehicle- and LY4-treated animals (n = 21 and 15). p was obtained by Mann-Whitney test. Figure 4B Is a graph showing quantification of circulating tumor cells / ml blood by HER2 staining of cell spindles. Figure 4C The images and graphs shown depict P-Rb per lung section / animal + Percentage of micrometastases (n = 4 and 6). Figure 4D The image shows 100% photoconversion of ZR75.1-H2B-Dendra from green fluorescence to red fluorescence on day 0 after implantation in 3D Matrigel. In Figure 4E , ZR75.1-H2B-Dendra photoconverted cells were seeded at low density (single cells) or high density. The graph shows the percentage of red label retention ± s.d. in cells seeded as single cells or at high density (n = 4). p was obtained by Student's t-test. Figure 4F 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 of viable colonies measured on day 8 ± s.d (n = 4). p was obtained by Student's t-test.

[0018] Figures 5A - 5C Confirms that inhibition of PERK is upregulated in Her2 + cells. Figure 5A Shows that PERK (EIF2AK3) is upregulated in a subset of HER2 + breast cancer patients. TCGA breast cancer data for HER2 + cases (58 tumors) were analyzed using cBioPortal. Figure 5B Shows representative images of carmine staining of whole mount FVB normal mammary glands compared to whole mount MMTV-neu mammary glands treated with vehicle and LY4. Figure 5C Shows quantification of histological architecture (from normal empty ducts to DCIS-like breast intraepithelial neoplasia), top image of H&E-stained breast sections, and higher magnifications below.

[0019] Figures 6A - 6C Shows that the PERK inhibitor LY4 causes mammary "normalization" in the MMTV-HER2 + breast cancer model. Figure 6A Shows representative images of carmine-stained whole mount mammary glands and H&E-stained breast sections from vehicle- and LY4-treated animals. Scale bar, 100 μm. Figure 6BShows the quantification (empty ducts e.d., occluded ducts o.d., obstructive hyperplasia o.h., and DCIS-like mammary intraepithelial neoplasia M.I.N) ± s.e.m. of histological structures found in H&E-stained mammary gland sections (n = 50 / animal, animal n = 13) in vehicle- and LY4-treated animals. Statistical significance (p) was calculated by 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 mammary gland sections. The figure shows CK8 / 18 + and SMA + scores of the structures, n = 12. p was obtained by Mann-Whitney test. Scale bar, 75 μm.

[0020] Figures 7A - 7F Shows the effect of LY4 treatment on P-PERK levels, P-histone H3 levels, and tumor size. Figure 7A Is a western blot for determining P-PERK levels in MMTV-neu tumor lysates from vehicle- and LY4-treated animals. Figure 7B Shows the tumor volumes (mm 3 ) of females from vehicle (upper) and LY4-treated (lower). Each line represents a tumor. Figure 7C Shows the percentage of tumor size reduction in LY4-treated females, which shows tumor shrinkage. Each line represents a tumor and an animal. Figure 7D Shows IHC of P-histone H3 in mammary tumor sections, representative images and quantification (right panel). p was obtained by Mann-Whitney test. In Figure 7E , ZR75.1 cells overexpressing HER2 were seeded on Matrigel, and after alveoli were established (day 10), the wells were treated with vehicle (control) or LY4 (2 μM) for 10 days. The figure shows the percentage of cleaved caspase-3 positive cells ± s.d. in each alveolus (n = 20). p was obtained by Student's t-test. In Figure 7F , MCF10A-HER2 cells were seeded on Matrigel, and after alveoli were established (day 4), the wells were treated with vehicle (control) or LY4 (2 μM) for 10 days. The figure shows the percentage of P-histone H3 positive cells ± s.d. in each alveolus (n = 20). p was obtained by Student's t-test.

[0021] Figures 8A - 8D Shows that inhibition of PERK impairs tumor growth in MMTV-HER2 + females. In Figure 8AIn this study, vehicle or LY4 (50 mpk) was injected daily into MMTV-neu female mice (24 to 32 weeks old) with palpable tumors for 2 weeks. The figure shows the percentage change in tumor size ± s.d. in vehicle- and LY4-treated animals (n = 16). p values were determined by Mann-Whitney test. Figure 8B is a graph showing the final tumor volume (mm 3 ). Whiskers represent the minimum and maximum values of the data (n = 16). p values were determined by Mann-Whitney test. Figure 8C Shows representative IHC of TUNEL staining to measure the level of apoptosis in tumor sections. Scale bars, 10 and 50 μm. Graph, percentage of TUNEL-positive cells in vehicle- and LY4-treated tumor sections (n = 5). p values were determined by Mann-Whitney test. In Figure 8D this study, HER2 + MCF10A-HER2 or SKBR3 cells were seeded on Matrigel, and after acini formation (day 4), the wells were treated with vehicle (control) or LY4 (2 μM) for 10 days. The figure shows the percentage ± s.d. of cleaved caspase-3-positive cells in each acinus (n = 20). p values were determined by Student's t test. Representative confocal images of MCF10A-HER2 acini stained for cleaved caspase-3.

[0022] Figures 9A - 9F Shows that treatment with LY4 decreased the levels of phosphorylated HER2 and downstream signaling pathways. Figure 9A Shows representative images of IHC for P-HER2, P-PERK, and P-EIF2α in MMTV-HER2 breast tumor sections. Note that the P-HER2-positive margins overlap with the staining of P-PERK and P-EIF2α. Scale bar, 100 μm. Figure 9B Shows hierarchical clustering of high-throughput targeted gene expression (columns) of single cells (primary breast tumors) (rows) from MMTV-HER2 females. Figure 9C Shows representative P-HER2 and total HER2 IHC staining in vehicle- and LY4-treated breast tumors. The figure shows the P-HER2 scores in vehicle- and LY-treated tumors. Quantification of P-HER2 levels in tumor sections by IHC intensity and area scoring (n = 11) (see Figure 10A ). Scale bar, 50 μm. p values were determined by Mann-Whitney test. In Figure 9DIn this experiment, MCF10A-HER2 cells were starved overnight and treated with + / - LY4 (2 μM), and then + / - EGF (100 ng / ml) was added for 15 minutes before collection. The levels of P-HER2, P-EGFR, P-AKT, P-S6, and P-ERK, as well as total HER2 and EGFR, were evaluated by Western blotting. GAPDH and β-TUB were used as loading controls. Representative blots of the three are shown. Densitometric analysis of P-HER2 (n = 3) ± s.d. p was obtained by Student's t-test. In Figure 9E In Figure 9D MCF10A-HER2 cells were treated as shown in Figure 9F and a surface receptor biotinylation assay was performed. The surface levels of total HER2 and P-HER2 were evaluated. Densitometry of P-HER2 is shown. In Figure 9D MCF10A-HER2 cells were treated as shown in

[0023] Figures 10A - 10C and a reversible surface receptor biotinylation assay was performed. The endocytic levels of total HER2 and P-HER2 were evaluated. One of the two experiments is shown. Figure 10A Quantification of the P-HER2 level in MCF10A-HER2 cells is shown. Figure 10B A scoring system for quantifying the P-HER2 level in breast tumor sections is shown. The IHCP-HER2 positive area was multiplied by its intensity score according to the determined score shown in these representative images. Scale bar, 100 μm. In Figure 10C The extract input used in the surface biotinylation assay is shown.

[0024] Figures 11A - 11E It is shown that inhibiting PERK after inhibiting CDK4 / 6 enhanced the anti-metastatic effect of LY4. Figure 11A is a schematic diagram of an in vivo experiment designed to evaluate the sequential treatment effects of abemaciclib and LY4 in a + MMTV-neu / HER2 + female mouse model. MMTV-neu / HER2 Figure 11BA series of fluorescence IHC of HER2, Ki67 (proliferation), and GADD34 (ER stress) were performed on tumor sections. Scale bar, 100 μm. Arrows indicate high fluorescence. Figure 11C The figure shows the number of macrometastases (>100 cells) detected by H&E staining and quantified in 5 lung sections / animal (n = 8). p was determined by Mann-Whitney test. Figure 11D The figure shows the number of micrometastases (2 - 100 cells) detected by IHC staining using anti-HER2 antibody and quantified in each lung section / animal ± s.d. (n = 8). p was determined by Matt-Whitney test. Figure 11E The figure shows the number of isolated disseminated tumor cells detected by classifying HER2 IHC staining as Ki67+ or Ki67 - and quantified in each lung section ± s.d. (n = 8). p was determined by Matt-Whitney test.

[0025] Figures 12A - 12G Shows the proposed monotherapy or combination therapy including the use of LY4, and experiments showed that treating melanoma cells with the CDK4 / 6 inhibitor abemaciclib in combination with LY4 differentially affected in vitro cell viability in 2D and 3D cultures. Figure 12A Is a schematic diagram of the combination principle of abemaciclib and LY4. Figure 12B Is a bar graph showing the results of treating Braf-mutated melanoma cells (WM35) with 0 nM, 10 nM, or 50 nM abemaciclib in vitro for 1 week and then treating with 2 μM LY4 for 48 hours. Figure 12C Includes cell images stained with DAPI after pretreatment with abemaciclib for 1 week and then treatment with 2 μM LY4. 5,000 cells were seeded on Matrigel. In Figures 12D - 12E WM35 melanoma cells were pretreated with abemaciclib for 5 weeks and then treated with a complete medium solution of LY4 and abemaciclib. Cells were stained with trypan blue to identify live cells. Figure 12D Is a figure showing abemaciclib-sensitive cells. Figure 12E Is a figure showing abemaciclib-resistant cells. Figure 12F Shows cell images stained with DAPI after pretreatment with abemaciclib for 5 weeks and co-treatment with 2 μM LY4 and abemaciclib. 1,000 cells were seeded on Matrigel. Figure 12GIt was shown that when growth arrest was induced by abemaciclib, cells upregulated the PERK target (GADD34), which may explain why the cells were sensitive to LY4. WM35 melanoma cells that were naïve or resistant (R) to abemaciclib were treated in cultures with vehicle (-) or 150 and 300 nM abemaciclib for 24 hours. Cells were then lysed and the expression of GADD34 was detected by Western blot. Tubulin expression was used as a loading control. Note that in naïve cells, GADD34 was upregulated, suggesting activation of PERK. Resistant cells appeared to show higher levels of GADD34, which did not change or decrease upon additional abemaciclib treatment.

[0026] Figures 13A - 13C The effects of BMP7-F9 on the ERK / p38 activity ratio and various mRNAs associated with quiescence signature genes were confirmed. Figure 13A It was shown that treatment with BMP7-F9 at 2 ng / ml, 5 ng / ml, and 10 ng / ml (second, third, and fourth grey columns respectively: control is the first black column) decreased the ERK / p38 activity ratio compared to control, as determined by Western blot in HEp3 HNSCC cells. The effect on the ERK / p38 activity ratio was observed at 2 - 6 and 24 hours (columns in the second to fourth groups). BMP7 (first column group) stimulated ERK activity within the first 30 minutes. Figure 13B It was shown that treatment with BMP7-F9 induced DEC2, p53, and p27 mRNAs encoding quiescence signature genes (10 ng / ml BMP7-F9, 24 hours). Figure 13C It was shown that treatment of the same cells with BMP7-F9 induced nuclear accumulation of NR2F1, a potential transcription factor that induces quiescence, as measured by immunofluorescence (10 ng / ml, 24 hours). Arrows indicate NR2F1 fluorescence. Figure 13A and Figure 13B The differences in were calculated by Student's t-test to give p < 0.05. These data support the hypothesis that BMP7-F9 is a strong inducer of quiescence genes that have been found to be upregulated in spontaneous quiescent DCCs or induced in the bone marrow via reprogramming or TGFβ2 signaling.

[0027] Figures 14A - 14E It was shown how BMP7-F9 induced growth arrest of T-HEp3 cells in vitro and in vivo. Figure 14A It was shown that treatment of T-HEp3 cells with BMP7-F9 inhibited their proliferation in vitro for 48 hours, as determined by CellTiter-Blue assay (RFU, relative fluorescence units). Figure 14B For Figures 14C - 14DSchematic of the in vivo experimental method used. T-HEp3 cells were pretreated with BMP7-F9 in vitro for 24 hours and then inoculated on the chorioallantoic membrane of chicken embryos (“CAM”) ( Figure 14C ), where they were treated in vivo with either vehicle or BMP7-F9 (50 ng / ml) daily, and then tumors were collected and the number of HEp3 HNSCC cells ( Figure 14D ) and the level of P-H3 ( Figure 14E ) were quantified. Figure 14E The arrows in indicate overlapping P-H3 and DAPI fluorescence. These data support the hypothesis that the dormancy markers identified in Figure 13B are associated with in vitro and in vivo growth inhibition in short-term experiments in the CAM system.

[0028] Figures 15A - 15C Shows the evaluation of BMP7-F9 treatment in a disease mouse model. Figure 15A is a schematic of the in vivo experimental method used to evaluate the effect of BMP7-F9 on the onset of metastasis. HEp3-GFP HNSCC tumors grew to approximately 300 mm 3 , and then were treated with 50 μg / kg of BMP7-F9 in a neo-adjuvant setting until the tumors were approximately 600 mm 3 . The tumors were then resected surgically. One to two days after surgery, BMP7-F9 adjuvant treatment was continued for an additional 3, 4, or 6 weeks. The animals were then euthanized, and the DCC burden in the lungs was scored using a fluorescence microscope. Figure 15B Shows that BMP7 limits the progression 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 the incidence of DCC were scored. In Figure 15C , mice were treated as in Figure 15A , except that the adjuvant treatment was 4 weeks instead. The number of GFP-positive cells in the isolated lungs was scored after treatment. This is a measure of the DCC burden in the lungs, which was significantly reduced by BMP7-F9 treatment. Note that the median DCC burden decreased by one log, and BMP-7 clearly cured DCC in 3 out of 7 animals. Detailed Description of the Invention

[0030] The present disclosure relates to methods of treating minimal residual cancer in a subject. One aspect of the present disclosure relates to a method of treating minimal residual cancer in a subject. The method involves contacting disseminated cancer cells (DCCs) in the subject with a bone morphogenetic protein 7 (BMP7)-derived protein. Contacting the disseminated cancer cells (DCCs) in the subject with the bone morphogenetic protein 7 (BMP7)-derived protein can induce or maintain dormancy in the contacted DCCs of the subject, thereby treating the minimal residual cancer of the subject.

[0031] As used herein, the phrase “minimal residual cancer” includes such a situation or condition where, by standard radiological and histological criteria, there is a lack of evidence of cancer in a subject, but where the subject actually has residual cancer cells (i.e., DCCs) in the blood (such as CTCs) or bone marrow or lymph nodes (such as DTCs). Minimal residual cancer can occur after treating cancer with chemotherapy, surgery, and / or radiotherapy. Standard radiological and histological detection methods can include, for example, imaging tests (X-rays, ultrasound, MRI); blood or immunochemical tests for known tumor markers or circulating tumor markers such as PSA; testing known tumor markers in biopsy or cytology specimens to assess, for example, the number of tumor cells present or the relative rarity of such cells.

[0032] It is well known in the art that tumor cells can spread early from primary tumors in the form of CTCs and DTCs. Indeed, DTCs have been identified in subjects who had no evidence of disease after tumor surgery. In rare cases where a cancer history fails to rule out an organ donation, donor-derived metastases have occurred in recipients even when the donor had been disease-free for up to 30 years (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 content of which is incorporated herein by reference).

[0033] Phylogenetic and whole-genome sequencing of metastatic tumors within individual patients has revealed the spread of primary tumors to metastases and metastases to metastases, providing evidence that the continuous / linear growth model cannot explain late (>10 years) recurrences in individual patients (Gundem et al., “The Evolutionary History of Lethal Metastatic Prostate Cancer,” Nature 520:353-357 (2015) and Naxerova et al., “Using Tumour Phylogenetics to Identify the Roots of Metastasis in Humans,” Nature Reviews Clinical Oncology 12:258-272 (2015), the entire contents of which are incorporated herein by reference).

[0034] Single-cell CTC analysis has also shown genetic lineage associations 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), the entire contents of which are incorporated herein by reference).

[0035] In addition, in humans, CTCs / DTCs are not associated with the stage or size of the primary cancer (Krishnamurthy et al., “Detection of Minimal Residual Disease in Blood and Bone Marrow in EarlyStage Breast Cancer,” Cancer 116(14):3330-3337(2010), the entire content of which is incorporated herein by reference). Instead, CTCs and DTCs are thought to retain the ability to form metastatic / recurrent disease. In particular, the detection of CTCs and DTCs has been shown to predict metastasis and recurrence in breast and prostate cancer (Braun et al., “A Pooled Analysis of BoneMarrow Micrometastasis in Breast Cancer,” NEJM 353:793-802(2005); Hayes et al., “Circulating Tumor Cells at Each Follow-up Time Point During Therapy ofMetastatic Breast Cancer Patients Predict Progression-Free and OverallSurvival,” Clin.Cancer Res. 12(14):4218-4224(2006); and de Bono et al., “CirculatingTumor Cells Predict Survival Benefit from Treatment in Metastatic Castration-Resistant Prostate Cancer,” Clin.Cancer Res. 14:6302-6309(2008), the entire content of which is incorporated herein by reference).

[0036] Metastasis is thought to be caused by proliferating DCCs and dormant DCCs that have undergone reactivation. Given that patients can develop metastatic lesions several years after tumor resection, dormant DCCs are thought to be a major factor contributing to late recurrence of cancer. As used herein, the term “dormancy” refers to a temporary mitotic and growth arrest, which is defined as cellular quiescence, in which intrinsic and / or extrinsic mechanisms drive individual DCCs or small populations of DCCs into a resting state (reversible growth arrest). A second type of dormant lesion is angiogenic dormancy, in which the balance between dividing cells and cells that die due to poor angiogenesis keeps the tumor mass constant. A third type is immune-mediated dormancy, in which the immune system keeps the proliferating tumor mass constant through continuous cytotoxic activity that continuously reduces the number of growing cancer cells (see, e.g., Sosa et al., “Mechanisms of Disseminated Cancer Cell Dormancy: An Awakening Field,” Nat. Rev. Cancer 14(9):611-622 (2014), the entire content of which is incorporated herein by reference). Dormant cells may be derived from established primary tumors, secondary tumors, and / or pre-invasive lesions.

[0037] In one embodiment of the methods disclosed herein, the DCCs contacted are dormant cancer cells, meaning that the cancer cells are undergoing a temporary mitotic / growth arrest or senescence-like behavior.

[0038] Hematopoietic lineage cells are eliminated by negative selection, and then DCCs in bone marrow aspirates can be detected by positive staining for EpCAM or CK8 / 18. Under combined conditions, cells can be stained for dormancy markers to determine whether they are in a proliferative or dormant state. The latter can be done after fixation. For whole-genome or whole-transcriptome analysis, EpCAM-positive DCCs from bone marrow are isolated live and subjected to whole-genome or transcriptome analysis ( 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), the entire content of which is incorporated herein by reference).

[0039] The methods described herein can further involve detecting the presence of DCCs in the subject prior to the contact. As shown in Table 1 below, quiescent DCCs can be identified because they are phenotypically distinguishable from other cell types (Sosa et al., “Mechanisms of Disseminated Cancer Cell Dormancy: An Awakening Field,” Nat. Rev. Cancer 14(9):611-622 (2014), the entire content of which is incorporated herein by reference).

[0040] Table 1. DCC Markers

[0041]

[0042] DTCs have been identified in the bone marrow of 13 - 72% of prostate cancer patients preoperatively and 20 - 57% of patients without evidence of disease more than 5 years postoperatively (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), the entire content of which is incorporated herein by reference). Detection of DTCs can predict recurrence in patients with clinical dormancy.

[0043] As used herein, the phrase “clinical dormancy” refers to a long clinically disease-free period (e.g., greater than 5 years) between removal of the primary tumor and disease recurrence. Clinical dormancy is common in prostate cancer, breast cancer, esophageal cancer, renal 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 Discov. Today Technol. 11:41-47 (2014); Gelao et al., “Tumour Dormancy and Clinical Implications in Breast Cancer,” Ecancermedicalscience 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), the entire contents of which are incorporated herein by reference), and reside in distant organs, including bone, lymph nodes, liver, and lung, where they can remain dormant for a long period (e.g., greater than 10 years) until clinical metastasis occurs in some patients.

[0044] In some embodiments of practicing the methods described herein, the subject has been diagnosed with CTCs.

[0045] In some embodiments of practicing the methods described herein, the subject has been diagnosed with DTCs and / or non-metastatic cancer.

[0046] As used herein, a "subject" is, for example, a patient such as a cancer patient, and includes any animal, but preferably a mammal. In one embodiment, the subject is a human subject. Suitable human subjects include, but are not limited to, children, adults, and elderly subjects who have been diagnosed with disseminated cancer cells and / or non-metastatic cancer.

[0047] In other embodiments, the subject can be a cow, sheep, pig, cat, horse, mouse, dog, rabbit, etc.

[0048] In the process of practicing the methods described herein, DCC in a subject is contacted to induce or maintain dormancy of DCC. This means establishing a persistent non-proliferative state in DCC or continuing a non-proliferative state in DCC.

[0049] In one embodiment, minimal residual cancer is treated in a subject that has been diagnosed with cancer. For example, but not limited to, the subject has been diagnosed with one or more of the following cancers: breast cancer, multiple myeloma, lung cancer, non-small cell lung cancer, brain cancer, cervical cancer, mantle cell lymphoma, leukemia, hepatocellular carcinoma, prostate cancer, urological and cutaneous melanoma, skin cancer, head and neck cancer, thyroid cancer, glioblastoma, neuroblastoma, and colorectal cancer.

[0050] Other cancers may also be suitable for treatment with the methods described herein.

[0051] In one embodiment, minimal residual cancer associated with or related to breast cancer is treated in a subject. The breast cancer can be selected from one or more of the following: invasive breast cancer, ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and inflammatory breast cancer.

[0052] Multiple molecular factors can be used to molecularly classify breast cancer, including the status of hormone receptors and human epidermal growth factor receptor 2 (HER2). HER2 and the basal-like group are the main 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), the entire content of which is incorporated herein by reference). In one embodiment, the breast cancer is HER2 + breast cancer.

[0053] In some embodiments of the methods described herein, the subject has undergone surgical resection to remove a tumor. For example, the subject may have undergone one or more of the following: mastectomy, prostatectomy, removal of a skin lesion, small bowel resection, gastrectomy, thoracotomy, adrenalectomy, appendectomy, colectomy, oophorectomy, thyroidectomy, hysterectomy, glossectomy, colorectal polyp resection, and colorectal resection.

[0054] In the methods described herein, disseminated cancer cells (DCCs) in a subject are contacted with a bone morphogenetic protein 7 (BMP7)-derived protein. BMP7 is a member of the TGFβ superfamily, which is secreted from bone marrow stromal osteoblasts and may affect the microenvironment of DCCs / DTCs. BMP7 plays a key role in the transformation of mesenchymal cells into bone and cartilage and has been shown to reversibly induce the regression of 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), the entire content of which is incorporated herein by reference). Pro-BMP7 is an intermediate between pro-BMP7 and mature BMP7 and is generated by proteolytic processing of the pro-protein, which generates the subunits of the mature homodimer.

[0055] Human BMP7 protein is a secreted signaling molecule in the TGF-β superfamily that was initially identified for its ability to induce bone formation but was later recognized as a multifunctional cytokine that mediates the growth and differentiation of many different cell types. The human BMP7 protein is expressed in cells as a precursor protein consisting of 292 amino acids, and the mature, biologically active BMP7 is generated by proteolytic removal of the signal peptide and pro-peptide. The amino acid sequence of wild-type human BMP7 protein, including the signal peptide (first 29 amino acids), pro-domain, and mature peptide (bold), is represented as SEQ ID NO: 1 and is shown below:

[0056]

[0057] One of ordinary skill in the art will understand that the signal peptide can be removed by proteolytic cleavage, thereby generating the intact pro-domain / mature peptide, which is referred to as pro-BMP7.

[0058] Wild-type human mature BMP7 is a dimer composed of two glycosylated, 139-amino acid disulfide-linked, approximately 35 kDa homodimeric proteins. Each homodimeric protein has the amino acid sequence shown in SEQ ID NO: 2:

[0059]

[0060] Variants of the human BMP7 protein include variants of human mature BMP7 shown in SEQ ID NO: 2, with specific amino acid changes indicated in the consensus sequence shown in SEQ ID NO: 3:

[0061]

[0062] Compared to the wild-type mature human BMP7 protein, specific variants of the human mature BMP7 protein described herein have elevated specific activity, improved solubility characteristics, improved bioavailability, reduced binding to endogenous circulating inhibitors, and / or reduced EBF activity.

[0063] Suitable variants of the human BMP7 protein are selected from the group: 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).

[0064] Table 2. Exemplary Variants of Human BMP7

[0065]

[0066]

[0067] In one embodiment, the variant of BMP7 is selected from the group: Y65G / I86L / N110G / Y128W and Y65G / I86L / F93V / N110G / Y128W.

[0068] In one embodiment, the BMP7 derivative is a modified BMP7 variant of pro-BMP7. The modified variant of pro-BMP7 can contain amino acid substitutions at amino acid positions corresponding to the mature protein domain of BMP7. The modified variant of pro-BMP7 can be processed into a mature BMP7-derived protein. Suitable variants of pro-BMP7 comprising a pro-domain fused to the N-terminus of the human mature BMP7 protein variant are 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, as shown in Table 3.

[0069] Table 3. Exemplary Variants of Human Pro-BMP7

[0070]

[0071]

[0072]

[0073] Suitable BMP7 derivative proteins for use in the methods described herein include variants of human pro-BMP7 (i.e., SEQ ID NO:1).

[0074] In one embodiment, the BMP7-derived protein is a mature BMP7 protein that has enhanced bioactivity (e.g., up to greater than 50-fold or more bioactivity) and biophysical properties (e.g., enhanced solubility and stability) compared to the mature wild-type BMP7 protein.

[0075] In another embodiment, the BMP7 derivative is BMP7-F9 (SEQ ID NO:8).

[0076] The wild-type BMP7 protein or its variants mentioned herein, including the SEQ ID NOs. mentioned, refer to homodimers in which each monomer subunit has a defined sequence. For example, reference to BMP7-F9 (SEQ ID NO:8) refers to such a homodimer in which each monomer subunit has the sequence shown in SEQ ID NO:8 and the subunits are linked by one or more disulfide bonds.

[0077] For the functional assays described herein, treating or administering with a specific pro-BMP7 protein or its variant refers to treating or administering with a homodimer of a specific mature BMP7, which is the wild-type or its variant, and the homodimer is typically in a non-covalent complex with the wild-type human pro-domain.

[0078] According to the methods described herein, exposure can be effected by administering the BMP7-derived protein to a subject.

[0079] The effect of BMP7 on a subject may depend on bone morphogenetic protein receptor 2 (BMPR2), the expression of which has been shown to be negatively 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 content of which is incorporated herein by reference). Thus, in one embodiment, the DCC for exposure in a subject is bone morphogenetic protein receptor positive (BMPR + ).

[0080] The methods described herein can further comprise administering to the subject a chemotherapeutic agent, an immunotherapeutic agent, an epigenetic agent, or ionizing radiation.

[0081] As used herein, the term “chemotherapeutic agent” refers to a synthetic, biological, or semi-synthetic compound that is not an enzyme and that can kill cancer cells or inhibit the growth of cancer cells with less effect on non-cancerous cells. Any suitable chemotherapeutic agent can be used.

[0082] 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-fluorouracil, capecitabine, tegafur, tegafur-uracil, floxuridine, 5-fluorodeoxyuridine, and S-1. Exemplary platinum drugs include, but are not limited to, cisplatin, carboplatin, oxaliplatin, and nedaplatin.

[0083] Other 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, dacarbazine, estramustine, streptozotocin, and temozolomide), vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine), podophyllotoxins (e.g., etoposide and teniposide), antibiotics (e.g., bleomycin, actinomycin, mitomycin, and valrubicin), and camptothecin analogs (e.g., irinotecan or topotecan).

[0084] In some embodiments, the chemotherapeutic agent is an anti-HER2 chemotherapeutic agent selected from trastuzumab and lapatinib. 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.

[0085] 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, an immunotherapeutic agent can stimulate the immune system to more effectively target cancer cells. Suitable immunotherapeutic agents can be selected from immune checkpoint inhibitors, interferons, and tumor vaccines.

[0086] Immune checkpoint inhibitors are compounds that inhibit the participation 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).

[0087] Interferons (“IFN”) are a family of cytokines that protect against disease through direct effects on target cells and by activating the immune response. IFN can be produced by and act on tumor cells and immune cells. Type I IFN includes IFNα proteins, IFNβ, IFNε, IFNκ, and IFNω. Type I IFN is known to mediate antitumor effects against a variety of malignancies (Moschos et al., “Interferons in the Treatment of Solid Tumors,” Cancer Treat. Res. 126:207–241 (2005), the entire content of which is incorporated herein by reference).

[0088] As used herein, the term "tumor vaccine" refers to a composition that stimulates an immune response against a tumor or cancer cells in a subject. Tumor vaccines typically consist of a source of cancer-related substances or cells (antigens) that may be autologous (from the subject's own body) or allogeneic (from another subject) to the subject, as well as other components (such as adjuvants) that further stimulate and enhance the immune response against the antigen. Tumor vaccines can result in the stimulation of the subject's immune system to produce antibodies against one or several specific antigens, and / or the production of cytotoxic T cells to attack cancer cells bearing those antigens.

[0089] As used herein, the term "epigenetic agent" refers to an agent that, upon contact with or after contact with such an agent, or upon administration or after administration of such an agent, can alter the epigenetic state (e.g., methylation state) of cellular DNA.

[0090] Suitable epigenetic agents can be selected from, for example, histone deacetylase ("HDAC") inhibitors, 5-azacytidine, retinoic acid, arsenic trioxide, Zeste 2 polycomb repressive complex 2 subunit enhancer (EZH2) inhibitors, bromodomain ("BRD") inhibitors, and derivatives thereof.

[0091] Exemplary HDAC inhibitors include, but are not limited to, trichostatin A, aspergillic acid, benzamide, phenylbutyrate, valproic acid, vorinostat, belinostat, LAQ824, Panobinostat, entinostat, CI994, and mocetinostat.

[0092] 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), the entire content of which is incorporated herein by reference).

[0093] 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,” Curis 8(5):e620 (2016), the entire content of which is incorporated herein by reference).

[0094] Other exemplary epigenetic agents include DNA methyltransferase (DNMT) inhibitors, including, but not limited to, azacitidine and decitabine.

[0095] The DCC / DTC microenvironment plays a key role in enhancing dormancy. Nuclear receptor subfamily 2F member 1 (NR2F1) is a nuclear hormone receptor and transcriptional regulator and is a key node in the transcriptional factor network that constitutes the characteristics of tumor cell dormancy. When applied to the gene expression profiles of patients with estrogen receptor-positive (ER + ) breast cancer, this signature has been shown to predict a longer metastasis-free period (Kim et al., “Dormancy Signatures and Metastasis in Estrogen Receptor Positive and Negative Breast Cancer,” PloS One 7:e35569 (2012), the entire content of which is incorporated herein by reference). This dormancy signature has also been found in dormant DTCs of prostate cancer patients who have been asymptomatic for 7 - 18 years (Sosa et al., “NR2F1 Controls Tumour 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), the entire content of which is incorporated herein by reference), highlighting its relevance to human disease.

[0096] In patient-derived xenograft (PDX) models of head and neck squamous cell carcinoma (HNSCC), NR2F1 has been shown to be upregulated after tumor surgery 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), the entire content of which is incorporated herein by reference). The plasticity of NR2F1 expression suggests that changes in the epigenome of residual tumor cells may be controlled by external and internal signals and determine the fate of DCC. NR2F1 has been shown to potentially limit the reprogramming of induced pluripotent stem cells (iPS) by regulating chromatin reprogramming (Onder et al., “Chromatin Modifying Enzymes as Modulators of Reprogramming,” Nature 483(7391):598-602 (2012), the entire content of which is incorporated herein by reference). NR2F1 is also key to maintaining global inhibitory chromatin in dormant tumor cells while allowing for an active chromatin state at the promoters of specific dormancy genes, including its own promoter (Sosa et al., “NR2F1 Controls Tumour Cell Dormancy via SOX9- and RARbeta-Driven Quiescence Programmes,” Nat. Commun. 6:6170 (2015), the entire content of which is incorporated herein by reference), highlighting the existence of a well-designed epigenetic program regulated by NR2F1 and microenvironmental cues that leads to tumor cell dormancy. In one embodiment, the DTC is NR2F1 + .

[0097] It has been shown that DCC can express high levels of PERK pathway activation (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), the entire contents of which are incorporated herein by reference), which mediates the ISR. ISR signaling through PERK and EIF2α phosphorylation results in a decrease in global translation, as well as an increase in gene-specific translation, oxidative stress, and ROS production, protein degradation, RNA degradation, autophagy, and lipid biosynthesis, which may contribute to the survival of tumor cells.

[0098] Another aspect relates to a method of treating minimal residual cancer in a subject, the method comprising contacting disseminated cancer cells (DCC) in the subject with a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor selected from LY2, LY3, and LY4, wherein the contacting eradicates the DCC in the subject to treat minimal residual cancer in the subject.

[0099] In one embodiment, the DCC has phosphorylated PERK activity. Thus, the method may further comprise contacting the DCC in the subject with a PERK inhibitor, a MEK inhibitor, a CDK4 / 6 inhibitor, or any combination thereof.

[0100] According to one embodiment of the methods described herein, the contacting can be effected by administering a PERK inhibitor to the subject.

[0101] In one embodiment, the PERK inhibitor is a compound of formula (I)

[0102]

[0103] wherein R is selected from the group consisting of, or a pharmaceutically acceptable salt thereof:

[0104]

[0105] X is CH or N;

[0106] R 1 is hydrogen or a halogen (e.g., fluorine); and

[0107] R 2 is a C1-C3 alkyl group.

[0108] In another embodiment, the PERK inhibitor is a compound represented by formula (Ia)

[0109]

[0110] wherein R is selected from the group consisting of, or a pharmaceutically acceptable salt thereof:

[0111]

[0112] X is CH or N;

[0113] R 1 is hydrogen or a halogen (e.g., fluorine); and

[0114] R 2 is a C1-C3 alkyl group.

[0115] When the inhibitor is a compound represented by formula (I) or formula (Ia), R can be

[0116]

[0117] As used herein, the term "alkyl" refers to an aliphatic hydrocarbon group, which can be straight-chain or branched-chain and has from about 1 to about 6 carbon atoms or 1 to about 3 carbon atoms in the chain (or is represented as "C n -C n ", where n is the numerical range of carbon atoms). Branched-chain means that one or more lower alkyl groups such as methyl, ethyl, or propyl are attached to the linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, and isopropyl.

[0118] The term "halogen" refers to fluorine, chlorine, bromine, or iodine. In one embodiment, the halogen is fluorine.

[0119] Where the context permits, the term "one or more compounds" and equivalent expressions refer to the compounds described herein, said expressions including prodrugs, pharmaceutically acceptable salts, oxides, and solvates, e.g., hydrates.

[0120] The compounds described herein may contain one or more asymmetric centers and thus may give rise to enantiomers, diastereomers, and other stereoisomeric forms. Each chiral center may be defined as (R)- or (S)- according to the absolute stereochemistry. The present invention is intended to embrace all such possible isomers, and mixtures thereof, including racemic and optically pure forms. The 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 should also be included.

[0121] References to a "compound" are intended to include salts, solvates, oxides, and inclusion compounds of the compound, and any stereoisomeric forms or mixtures of any such forms of the compound, in any proportion. Thus, according to some embodiments, the compounds described herein are provided in the form of salts, including in the context of pharmaceutical compositions, methods of treatment, and the compounds themselves.

[0122] The term "solvate" refers to a solid-state compound in which suitable solvent molecules are incorporated into the lattice. Suitable solvents for therapeutic administration are physiologically tolerable at the doses administered. Examples of suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is called a hydrate. Generally, solvates are formed by dissolving the compound in an appropriate solvent and separating the solvate by cooling or using an antisolvent. Solvates are usually dried under ambient conditions or by azeotropic distillation.

[0123] Inclusion compounds are described in Remington, The Science and Practice of Pharmacy, 19th Ed. 1:176 - 177 (1995), the entire content of which is incorporated herein by reference. The most commonly used inclusion compounds are those with cyclodextrins, and all natural and synthetic cyclodextrin complexes are specifically covered by the present invention.

[0124] The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases (including inorganic and organic acids and bases).

[0125] The term "pharmaceutically acceptable" means that within the scope of reasonable medical judgment, it is suitable for contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response, etc., and is commensurate with a reasonable benefit / risk ratio.

[0126] 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.

[0127] Table 4. Exemplary PERK inhibitors

[0128]

[0129] In some embodiments, contact is made with a PERK inhibitor that does not inhibit EIF2AK1, EIF2AK2, or EIF2AK4.

[0130] In one embodiment, the PERK inhibitor does not inhibit AXL. According to this embodiment, the PERK inhibitor is selected from LY3 and LY4.

[0131] In another embodiment, the PERK inhibitor does not inhibit Flt3, MNK2, or NTRK. According to this embodiment, the PERK inhibitor is LY4.

[0132] In one embodiment, contact is made by administering a MEK inhibitor to the subject. Exemplary MEK inhibitors are well known in the art and include, for example, PD184352, PD318088, PD98059, PD334581, 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 content of which is incorporated herein by reference).

[0133] In another embodiment, contact is made 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).

[0134] In one embodiment, the method may further involve selecting a subject without signs of disease prior to the contact. For example, the subject may be in cancer remission prior to the contact.

[0135] In practicing the methods described herein, minimal residual cancer is treated in a subject. Such treatment can include, but is not limited to, administering to a subject in need of treatment for minimal residual cancer one or more compounds effective to treat the disease (i.e., cancer or minimal residual cancer) in the subject.

[0136] In one embodiment, the methods of treatment described herein are carried out under conditions effective to induce dormancy in disseminated tumor cells (“DTCs”) and / or induce the death of dormant DTCs.

[0137] In practicing the methods of treatment described herein, administering a compound to a subject can involve administering a pharmaceutical composition comprising the compound (i.e., the BMP7-derived protein and PERK inhibitor described herein) in a therapeutically effective amount, which refers to the amount of the compound effective to treat the disorder and / or disease in the subject. Such amount will generally vary depending on a number of factors within the capabilities of a person of ordinary skill in the art. These include, but are not limited to, the particular subject, and the subject's age, weight, height, general physical condition, and medical history, the particular compound used, and the carrier in which it is formulated and the route of administration selected therefor; the time or duration of treatment; and the nature and severity of the disorder being treated.

[0138] Administration generally involves administering a pharmaceutically acceptable dosage form, which refers to the dosage forms of the compounds described herein and includes, for example, tablets, pills, powders, elixirs, syrups, liquid preparations, including suspensions, sprays, inhalants, tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories, and liquid preparations for injection, including liposomal formulations. Techniques and formulations can generally be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition, the entire contents of which are incorporated herein by reference.

[0139] In practicing the methods of treatment described herein, the drug (i.e., the BMP7-derived protein and PERK inhibitor described herein) can be included in any suitable carrier substance in any suitable amount. The drug can be present in an amount up to 99 weight % of the total weight of the composition. The composition can be provided in dosage forms suitable for the following routes: oral, parenteral (e.g., intravenous, intramuscular), rectal, dermal, nasal, vaginal, inhalation, transdermal (patch), or intraocular administration. Thus, the composition can be in the following dosage forms: for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drops, osmotic delivery devices, suppositories, enemas, injections, implants, sprays, or aerosols.

[0140] The pharmaceutical composition according to the present disclosure can be formulated to release the active drug in large amounts immediately after administration or at any predetermined time or time period after administration.

[0141] Controlled release formulations include (i) formulations that produce a substantially constant concentration of a drug in the body over a long period of time; (ii) formulations that produce a substantially constant concentration of a drug in the body over a long period of time after a predetermined lag time; (iii) formulations that maintain the drug effect over a predetermined period of time by maintaining a relatively constant effective drug level in the body and simultaneously minimizing adverse side effects associated with fluctuations in the plasma level of the active drug substance; (iv) formulations that localize the drug effect by, for example, spatially placing a controlled release composition near or inside a diseased cell, tissue, or organ; and (v) formulations that target the drug effect by delivering the drug to a specific target cell type using a carrier or chemical derivative.

[0142] Administration in the form of a controlled release formulation is preferred in situations where the drug has (i) a narrow therapeutic index (i.e., the difference between the plasma concentration that causes harmful side effects or toxic reactions and the plasma concentration of the drug that causes a therapeutic effect is small; generally, the therapeutic index (TI) is defined as the ratio of the median lethal dose (LD50) to the median effective dose (ED50)); (ii) a narrow gastrointestinal absorption window; (iii) a very short biological half-life such that frequent administration throughout the day is required to maintain the plasma level at a therapeutic level.

[0143] Any one of a variety of strategies can be employed to obtain controlled release in which the release rate of the drug under discussion is greater than the metabolic rate. Controlled release can be obtained by appropriately selecting various formulation parameters and ingredients, including, for example, various types of controlled release compositions and coatings. Thus, the drug is formulated with a suitable excipient into a pharmaceutical composition that will release the drug in a controlled manner after administration (single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes).

[0144] Thus, administration according to the method of the present disclosure can be carried out by oral administration, topical administration, transdermal administration, parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intraperitoneal administration, by intranasal instillation, by intracavitary or intravesical instillation, intraocular administration, intraarterial administration, intralesional administration, or by mucosal administration. The compound can be administered alone or in combination with a suitable pharmaceutical carrier and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.

[0145] The medicament (i.e., the BMP7 derivative protein and PERK inhibitor of the present disclosure) can be administered orally, for example, orally with an inert diluent, or with an absorbable edible carrier, or can be enclosed in a hard or soft shell capsule for oral administration, or can be compressed into tablets for oral administration, or can be mixed with food for oral administration. For oral therapeutic administration, the medicament can be combined with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, etc. Such compositions and formulations should contain at least 0.001% of the active compound. Of course, the percentage of the compound in these compositions can vary and can conveniently be between about 0.01% and about 10% by unit weight. The amount of the active compound in such therapeutically useful compositions enables a suitable dosage to be obtained. In one embodiment, the composition is prepared such that an oral dosage unit contains about 1 μg to 1 g of the active compound.

[0146] Tablets, capsules, etc. may also contain binders such as gum tragacanth, gum arabic, corn starch or gelatin; excipients such as dibasic calcium phosphate; disintegrants such as corn starch, potato starch, alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose or saccharin. When the dosage unit form is a capsule, it may also contain a liquid carrier such as a fatty oil in addition to the above types of materials.

[0147] A variety of other materials can be present as coatings or used to alter the physical form of the dosage unit. For example, tablets can be coated with shellac, sugar, or both. In addition to the active ingredient, syrups can also contain sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes, and flavoring agents such as cherry or orange flavoring agents.

[0148] The therapeutic agent can also be administered parenterally. Solutions or suspensions can be prepared in water in a suitable manner mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Exemplary oils are of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, or mineral oil. Generally, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycol, hyaluronic acid and its derivatives, carboxymethylcellulose and other soluble polysaccharide derivatives, or polyethylene glycol are preferred liquid carriers, especially for injectable solutions. Under ordinary storage and use conditions, these formulations contain preservatives to prevent the growth of microorganisms if they are not produced in a sterile manner.

[0149] 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 must flow to an extent that allows for easy injection. It must be stable under the conditions of manufacture and storage and must be protected from contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium that includes, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0150] The therapeutic agent can also be administered directly to the airway in the form of an aerosol. For use as an aerosol, a solution or suspension of the therapeutic agent can be packaged in a pressurized aerosol container together with a suitable propellant, such as hydrocarbon propellants like propane, butane, or isobutane together with conventional adjuvants. The therapeutic agent can also be administered in a non-pressurized form, such as in a nebulizer or atomizer.

[0151] In one embodiment, administration can increase the amount of detectable dormant DCC in a subject by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more.

[0152] In another embodiment, administration can decrease the amount of detectable DCC in a subject by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more.

[0153] In the context of the present disclosure, "treatment" refers to maintaining in a subject the absence of evidence of symptomatic disease (e.g., cancer).

[0154] In one embodiment, the terms "treating" or "treatment" specifically refer to eliminating minimal residual cancer in a subject. The term treatment includes inducing dormancy in DCC. The term treatment also includes eliminating dormant DCC in the subject. The term treatment also includes reducing the amount or quantity of detectable dormant DCC in a subject.

[0155] Another aspect of the present disclosure relates to a method of treating minimal residual cancer in a subject. The method involves contacting disseminated cancer cells (DCC) in the subject with a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor selected from LY2, LY3, and LY4, wherein the contacting eradicates the DCC in the subject to treat the minimal residual cancer in the subject.

[0156] As described above, the methods of the present disclosure are suitable for treating minimal residual cancer in a subject diagnosed with one or more of the following diseases: 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.

[0157] The cancer can be breast cancer selected from the following: invasive breast cancer, ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and inflammatory breast cancer.

[0158] In one embodiment, the breast cancer is HER2 + breast cancer.

[0159] In another embodiment, the subject has been diagnosed with disseminated tumor cells and / or non-metastatic cancer.

[0160] As described above, the methods of the present disclosure can further involve administering to the subject a chemotherapeutic agent, an immunotherapeutic agent, an epigenetic agent, or ionizing radiation.

[0161] When a chemotherapeutic agent is administered to the subject, the chemotherapeutic agent can be an anti-HER2 chemotherapeutic agent selected from trastuzumab and lapatinib . In another embodiment, the chemotherapeutic agent can be selected from anthracyclines, taxanes, kinase inhibitors, antibodies, fluoropyrimidines, and platinum drugs.

[0162] When an immunotherapeutic agent is administered to the subject, the immunotherapeutic agent is selected from immune checkpoint inhibitors, interferons, or tumor vaccines.

[0163] When an epigenetic agent is administered to the subject, the epigenetic agent can be selected from histone deacetylase (HDAC) inhibitors, 5-azacytidine, retinoic acid, arsenic trioxide, Zeste 2 polycomb repressive complex 2 subunit enhancer (“EZH2”) inhibitors, bromodomain (BRD) inhibitors, and derivatives thereof.

[0164] Contact can be carried out by administering the PERK inhibitor to the subject. Suitable PERK inhibitors have been described in detail above and include, but are not limited to, LY2, LY3, and LY4.

[0165] In one embodiment, the method further involves detecting the presence of DTC in the subject prior to the contact. As described in more detail above, the DTC can be NR2F1 + , having phosphorylated PERK activity and / or BMPR+ 。

[0166] The method can further involve contacting DCC / DTC in the subject with a BMP7-derived protein. In one embodiment, the DCC / DTC in the subject is contacted with a BMP7-derived protein by administering the BMP7-derived protein to the subject. Suitable BMP7-derived proteins are as described above. In one embodiment, the BMP7-derived protein is BMP7-F9.

[0167] In one embodiment, the PERK inhibitor does not inhibit EIF2AK1, EIF2AK2 or EIF2AK4.

[0168] As described above, the subject can be a mammal, preferably a human.

[0169] In one embodiment, the method can further involve selecting a subject without signs of disease prior to the contacting. For example, the subject can be in cancer remission prior to the contacting.

[0170] Another aspect of the disclosure relates to a method of treating advanced cancer in a subject. The method involves contacting disseminated cancer cells (DCC) in the subject with a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor selected from LY2, LY3, and LY4, wherein the contacting eradicates DTC in the subject to treat minimal residual cancer in the subject.

[0171] As used herein, the term "advanced cancer" refers to stage II cancer, stage III cancer, and / or stage IV cancer, or any cancer that has metastasized. It should be understood that the "advanced" nature of the cancer disease state can be determined by a physician.

[0172] 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.

[0173] In one embodiment, the cancer is breast cancer selected from the group consisting of invasive breast cancer, ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and inflammatory breast cancer. Breast cancer can be HER2 + breast cancer.

[0174] The method can further involve administering to the subject a chemotherapeutic agent, an immunotherapeutic agent, an epigenetic agent, or ionizing radiation. In one embodiment, the chemotherapeutic agent is selected from trastuzumab and lapatinib An anti-HER2 chemotherapeutic agent. In another embodiment, the chemotherapeutic agent is selected from anthracyclines, taxanes, kinase inhibitors, antibodies, fluoropyrimidines, and platinum drugs. The immunotherapeutic agent can be selected from immune checkpoint inhibitors, interferons, or tumor vaccines. The epigenetic agent can be selected from histone deacetylase (HDAC) inhibitors, 5-azacytidine, retinoic acid, arsenic trioxide, enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) inhibitors, bromodomain (BRD) inhibitors, and derivatives thereof.

[0175] In one embodiment, the contacting is performed by administering the PERK inhibitor to the subject.

[0176] The method can further involve detecting the presence of DCC / DTC in the subject prior to the contacting.

[0177] As described above, the DTC can be NR2F1 + or have phosphorylated PERK activity. Examples

[0178] Materials and methods for Examples 1 - 2 - 6

[0179] Reagents, cell culture, and treatment: EGF was obtained from PeproTech (Rocky Hill, NJ) and used at 100 ng / ml. Thapsigargin was purchased 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), the entire content of which is incorporated herein by reference). For 3D culture, MCF10A-HER2, SKBR3, and ZR75.1-H2B-Dendra2 cells were plated in growth factor-reduced Matrigel (Corning, Corning, NY) and grown 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), the entire content of which is incorporated herein by reference). When referring to “low density,” 3,500 cells / 8 wells were seeded, and for “high density,” 20,000 cells / 8 wells were seeded. Treatments with vehicle (DMSO) or LY4 (2 μM) were replaced every 24 hours in 2D culture and every 48 hours in 3D culture.

[0180] Mice, tumor growth, and tissue processing: The FVB / N-Tg(MMTVneu) mouse strain was obtained from The Jackson Laboratory (Sacramento, CA). These mice express an inactive form of neu (HER2) under the transcriptional control of the mouse mammary tumor virus promoter / enhancer. Prior to any experiments, females underwent one round of pregnancy and were allowed at least two weeks without lactation after weaning. Female mice aged 24-32 weeks were injected intraperitoneally with vehicle (90% corn oil, 10% ethanol) or LY4 (50 mpk) daily for two weeks. For combination therapy, female mice aged 24-32 weeks were treated by gavage with abemaciclib (50 mpk) daily for 4 weeks prior to starting the above treatment with LY4. Using the formula (Dxd 2) / 2 to measure the tumor volume, where D is the longest diameter and d is the shortest diameter. For CTC counting, the animals were anesthetized and whole blood was extracted by cardiac puncture. The mammary glands, lungs, and tumors were collected and fixed overnight in 10% buffered formalin before paraffin embedding. The bone marrow from both hind limbs was flushed with a 26G needle and further processed by Ficoll density gradient centrifugation. For CTC and for DTC detection in the bone marrow, mature hematopoietic cells in the tissues were first depleted by magnetic bead separation using anti-mouse antibodies (Miltenyi Biotec, San Diego, CA), and then fixed in formalin solution at 4 °C for 20 minutes.

[0181] Whole-mount mammary gland staining: The mammary glands fixed in 10% buffered formalin were incubated in carmine alum stain (carmine 0.2%, potassium aluminum sulfate 0.5%) (Sigma, St. Louis, MO) for 2 days. Then, they were dehydrated and transferred to methyl salicylate solution before imaging using a stereomicroscope.

[0182] 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), the entire content of which is incorporated herein by reference). Briefly, the slides were deparaffinized and rehydrated successively. Heat-induced antigen retrieval was performed in citrate buffer (10 mM, pH 6), EDTA buffer (1 mM, pH 8), or Tris / EDTA (pH 9). The slides were incubated in 0.1% Triton TM- Further permeabilized, blocked in -X100 and incubated overnight at 4°C with the primary antibodies at a dilution of 1:50 - 1:200. For IHC, additional steps of endogenous peroxidase and avidin / biotin quenching were performed prior to the 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), the entire content of which is incorporated herein by reference), P-EIF2A, cleaved Caspase 3, P-H3 (S10), P-HER2 (Y1221 / 1222) (Cell signaling, Danvers, MA), P-Rb (S249 / T252) (Santa Cruz, Dallas, TX), HER2 (Abcam, Cambridge, MA), HER2 (Millipore, Darmstadt, Germany), Ki67 (eBioscience and Abcam), cytokeratin mix (C11 and ck7, Abcam; AE1 and AE3, Millipore) and GADD34 (Santa Cruz). Next, the slides were incubated and fixed in the secondary antibody (Life Technologies, Norwalk, CT). For IHC, the sections were processed using the VectaStain ABC Elite kit (Vector Laboratories, Bulingame, CA) and the DAB substrate kit was used for peroxidase labeling (Vector Laboratories), and then the sections were mounted in VectaMount medium (Vector Laboratories). For IF, the sections were mounted in ProLong Gold Antifade aqueous medium (Thermo Fisher, Waltham, MA).

[0183] In the case of immunocytofluorescence, cytocentrifuge smears of fixed cells (100,000 - 200,000 cells / cytocentrifuge smear) were prepared by centrifuging cells at 500 rpm for 3 minutes on a multi-preparation slide, and the staining steps were carried out according to the instructions below starting from the permeabilization step. For the staining of 3D cultures, the acini were fixed in 4% PFA at 4 °C for 20 minutes, permeabilized with a 0.5% Triton TM -X100 PBS solution at room temperature for 20 minutes, washed in PBS-glycine, and then blocked with 10% normal goat serum at 37 °C for 1 hour, followed by immunofluorescence staining.

[0184] In Figure 10A the scoring of P-HER2 levels is explained. For the scoring of CK8 / 18 and SMA in mammary ducts, the expression of CK8 / 18 was evaluated as negative (0), low (1), or high (2) in 20 low-power fields per animal, and SMA was evaluated similarly, and the sum of the two scores was used as the final score (ranging from 0 to 4).

[0185] Microscopic observation: Images were captured using a Nikon Eclipse TS100 microscope, a Leica DM5500, or a confocal Leica SP5 multiphoton microscope.

[0186] TUNEL in situ cell death detection: The apoptosis level was evaluated using an in situ cell death detection kit AP (Roche, Basel, Switzerland). Paraffin sections from tumors were deparaffinized, rehydrated, and permeabilized in 0.2% TRITON TM -X100 phosphate-buffered saline (PBS) for 8 minutes. Then, the slides were washed and blocked with 20% normal goat serum at 37 °C for 1 hour. Then, the TUNEL reaction mixture was added and incubated at 37 °C for 1 hour. The reaction was terminated by incubation with buffer I (0.3 M sodium chloride, 30 mM sodium citrate). Subsequently, the slides were incubated with anti-fluorescein-AP antibody at 37 °C for 30 minutes. After washing 3 times in Tris-buffered saline (TBS), the slides were incubated in a 0.1% TWEEN TM -20 solution of alkaline phosphatase substrate at room temperature for 20 minutes. Finally, the slides were fixed using an aqueous mounting medium. The percentage of TUNEL-positive cells was calculated using Image J software (NIH).

[0187] Immunoblot analysis: As previously described, cells were lysed in RIPA buffer and proteins were analyzed by immunoblotting (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), the entire content of which is incorporated herein by reference). The membranes were blotted with the following additional antibodies: 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), the entire content of which is incorporated herein by reference), 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 in low-adhesion plates for 24 hours before collection.

[0188] Cell surface biotinylation and endocytosis assays: 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 h, then stimulated with + / - EGF (100 ng / ml) for 20'. Then, the cells were washed with ice-cold PBS and surface proteins were biotinylated at 4 °C for 30 min. After quenching, the cells were collected and lysed using RIPA buffer. The protein lysates were incubated with NeutrAvidin agarose beads and the 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), the entire content of which is incorporated herein by reference), the cells were treated similarly, but the cell surface proteins were biotinylated before treatment with EGF. After incubation at 37 °C with + / - EGF (100 ng / ml) for 20 min (to induce endocytosis), the cells were washed with ice-cold PBS and incubated with stripping 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) for 30'. To control the stripping efficiency, the cells were stripped without 37 °C incubation (t = 0). Cell lysates were prepared and processed as described above for the isolation of biotinylated proteins.

[0189] Single-cell targeted gene expression analysis: Primary tumors from 28-30-week-old female MMTV-neu were digested into single-cell suspensions with collagenase. Lungs from 15-30-week-old female MMTV-neu were digested into single-cell suspensions with collagenase and then resuspended in FACS buffer. The cells were then stained with anti-HER2-PE, anti-CD45-APC, and DAPI, and the HER2+ / CD45- cell population was sorted using a BD FACSAria sorter. The sorted cells were resuspended in medium at a concentration of 312,500 cells / ml, and then 80 μl was mixed with 20 μl of suspension reagent (C1 Fluidigm). Single-cell isolation was performed using a C1 Single-Cell Preamp IFC 10-17 μm. Pre-amplification was performed using Ambion's Single-Cell to CT qRT-PCR Kit and 20x TaqMan Gene Expression FAM-MGB Assays. The resulting cDNA was further diluted in C1 DNA Diluent 1 / 3, and gene expression analysis was performed using a 96.96 IFC (Fluidigm), Juno System Controller, and Biomark HD for high-throughput qPCR. qPCR reactions were performed using TaqMan Fast Advanced Master Mix. Analysis was performed using Fluidigm Real-Time PCR Analysis Software and the web-based tool Clustergrammer (Fernandez et al., “Clustergrammer, A Web-Based Heatmap Visualization and Analysis Tool for High-Dimensional Biological Data,” Sci. Data 4:1-12 (2017), the entire content of which is incorporated herein by reference) for hierarchical clustering heatmaps.

[0190] Biochemical assays: Recombinant human EIF2AK3 (PERK) catalytic domain (amino acids 536-1116; catalog number PV5107), GFP-eIF2α (catalog number PV4809) substrate, and Terbium-labeled phosphorylated eIF2α antibody (catalog number PR8956B) were purchased from Invitrogen (Carlsbad, CA). HIS-SUMO-GCN2 catalytic domain (amino acids 584-1019) was expressed and purified from Escherichia coli. The TR-FRET kinase assay was performed in a reaction buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl2, 1.0 mM EGTA, 0.01% Brij-35, and 100-200 nM GFP-eIF2α substrate in the absence or presence of inhibitors. The PERK assay contained 62.5 ng / ml of enzyme and 1.5 μM ATP (Km,app ~1.5 μM), and the GCN2 assay contained 3 nM enzyme and 90 μM ATP (K m,app ~200 μM). After addition of the test compound, the reaction was initiated by adding the enzyme and incubated for 45 minutes at room temperature. The reaction was terminated by adding EDTA to a final concentration of 10 mM, and a terbium-labeled phosphorylated eIF2α antibody was added at a final concentration of 2 nM and incubated for 90 minutes. In a Multilabel plate reader (PerkinElmer, Waltham, MA), the resulting fluorescence was monitored. The TR-FRET ratio and the resulting IC 50 value were determined from the fitted inhibition curve. Biochemical specificity assays were performed at Cerep (Redmond, WA) and DiscoverX (San Diego, CA).

[0191] Cell-based TR-FRET assay: Briefly, GripTite TM 293 cells (Invitrogen) expressing GFP-eIF2α were seeded at 10,000 cells per well in a 384-well plate and allowed to attach overnight. The cells were pretreated with the test compound for 1 hour. Tunicamycin (1 μM) was added to induce PERK activity, and the plate was incubated at 37 °C for 2 hours. The medium was removed, and the 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 inhibitor (Sigma catalog number P8340), phosphatase inhibitor (Sigma catalog number P2850), and 2 nM terbium-labeled anti-phosphorylated eIF2 antibody (Invitrogen catalog number PM4312I). The cell lysate was incubated for 2 hours at room temperature in the dark and in a Multilabel plate reader (PerkinElmer, Waltham, MA), the fluorescence was monitored. Using uninduced (100% inhibition) and induced (0% inhibition) wells as controls, the TR-FRET ratio and the resulting IC50 value were determined from the fitted inhibition curve.

[0192] ATF4-luc assay: 293 cells were transduced with a lentivirus expressing ATF4-luc (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 per well in poly D-lysine-coated 96-well plates and allowed to attach overnight. The cells were then pretreated with the test compounds for 30 minutes. Tunicamycin (2 μM) was added to induce ER stress, and the plates were incubated at 37 °C for 6 hours. The medium was then aspirated, and the cells were lysed in passive lysis buffer (Promega Cat#E194A) on a plate shaker for 5 minutes. Luciferase activity was monitored using luciferase assay reagent (Promega Cat#E1501) in a Wallac 1420 Victor2 TM Multilabel Counter (PerkinElmer, Waltham, MA), and the IC 50 values were determined from the resulting fitted inhibition curves using uninduced (100% inhibition) and induced (0% inhibition) wells as controls.

[0193] Cell viability assay: The growth of Hela, HT-1080, and Bx-PC-3 cells in 96-well plates was monitored in the absence or presence of a PERK inhibitor for 48, 72, or 96 hours. Cell viability was determined using CellTiter- Reagent (Promega, Madison, WI), and the IC 50 values were determined from the resulting fitted inhibition curves using untreated (0% inhibition) and 20 μM staurosporine-treated (100% inhibition) wells as controls.

[0194] Statistical analysis: All points represent independent biological samples, error bars represent standard deviation, and statistical significance was determined using the Mann-Whitney test with Graph Pad Prism software.

[0195] Example 2 – Quiescent HER2 + DTC exhibits an ER stress response

[0196] It has been shown that activation of the PERK pathway is a key effector of UPR-induced growth arrest and survival associated with the dormant phenotype (Brewer et al., “PERK Mediates Cell-Cycle Exit During the Mammalian Unfolded Protein Response,” Proc. Natl. Acad. Sci. U.S.A. 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), the entire contents of each of these references are incorporated herein by reference). In MMTV-HER2 animals, a higher percentage of mice develop lung metastases, which may be initiated by early DCC 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. U.S.A. 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), the entire contents of these references are incorporated herein by reference). Dormant DCC exhibits loss of E - cadherin and expression of Twist1 (Harper et al., "Mechanism of Early Dissemination and Metastasis in Her2 + "Mammary Cancer," Nature 540:588 - 592(2016), the entire contents of which are incorporated herein by reference), and E - cadherin - negative DCC in pancreatic cancer models has also been shown to be quiescent and to exhibit upregulation of CHOP (a PERK - induced gene) (Pommier et al., "Unresolved Endoplasmic Reticulum Stress Engenders Immune - Resistant, Latent Pancreatic Cancer Metastases," Science 360(6394):eaao4908(2018), the entire contents of which are incorporated herein by reference). To evaluate whether this same correlation between PERK pathway activation levels and cell cycle arrest exists in the MMTV - HER2 spontaneous metastasis model, two different approaches were used - high - resolution imaging using immunofluorescence (IF) and gene expression analysis at single - cell resolution of DCC and metastasis. IF of lung tissue sections from MMTV - HER2 animals carrying large tumors and thus carrying dormant and proliferative DCC was performed (Harper et al., "Mechanism of Early Dissemination and Metastasis in Her2 +“Mammary Cancer,” Nature 540:588 - 592(2016), the entire content of which is incorporated herein by reference). Next, tissues were co - stained to detect DCCs that were positive for HER2, Ki67 (as a marker of proliferation), and GADD34 (or PPP1r15A). GADD34 is a PERK - induced stress gene responsible for the programmed transition from translational repression (attributed to phosphorylation of eIF2α) to stress - induced gene expression (Novoa et al., “Stress - Induced Gene Expression Requires Programmed Recovery from Translational Repression,” EMBO J. 22:1180 - 7(2003), the entire content of which is incorporated herein by reference). Image analysis showed that HER2 + metastatic lesions or DCCs with a low proliferation index (ki67 low ) exhibited high levels of ER stress, as indicated by high levels of GADD34 expression ( Figure 1A , upper panels and graphs). On the other hand, highly proliferative DCCs or lesions showed very low levels of GADD34 staining ( Figure 1A , lower panels and graphs). The two markers, Ki67 and GADD34, were inversely correlated in 100% of the cells, which supports the use of GADD34 detection as a biomarker for UPR 高 , quiescent DCCs, and metastatic lesions.

[0197] Next, these correlations were evaluated in human breast metastatic lesions by testing 17 breast cancer metastases from different subtypes and sources (lymph node, lung, liver) (Table 5). Breast cancer metastases were stained for cytokeratin to identify metastatic lesions, Ki67, and GADD34. Compared to those in the mouse model, advanced human metastatic lesions showed a more heterogeneous staining pattern for both markers between different patients and between different regions of the same lesion. However, an inverse correlation independent of the type of metastasis was observed between the level of proliferation (Ki67) and ER stress activation (GADD34) ( Figure 1B ). This analysis confirmed the findings in the mouse model and confirmed that GADD34 may contribute to the identification of UPR 高 / quiescent tumor cells in metastatic sites.

[0198] Table 5. Human breast cancer metastasis samples

[0199]

[0200]

[0201] *High (H); Low (L); Medium (I)

[0202] By performing single-cell targeted gene expression analysis of DCC, micrometastasis, and macrometastasis in the lungs of MMTV-HER2 mice, markers of proliferation, quiescence, dormancy, and ER stress present in metastatic cells were evaluated. Lungs from MMTV-HER2 females were processed into single-cell suspensions, and HER2 + / CD45 - cells ( Figure 2A ) were sorted. Then, as Figure 2A shown, the sorted cells were processed using C1 (Fluidigm) technology for single-cell isolation, lysis, RT, and preamplification. This pipeline enables high-confidence (IF and molecular confirmation of HER2 + single cells) and high-quality isolation and processing of 255 individual DCCs and 90 primary tumor cells and their corresponding libraries. Next, high-throughput qPCR was used to analyze the expression of ER stress genes, cell cycle genes (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), the entire content of which is incorporated herein by reference; 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), the entire content of each of the aforementioned references is incorporated herein by reference) ( Figure 2B ). DCC gene expression at single-cell resolution revealed the presence of a subset of cells ( Figure 1C , Group 1, ~19% of DTCs) that showed strong upregulation of all tested ER stress genes (including PERK itself) (boxed with Fam123b-Ddit3) along with negative regulators of cell proliferation such as Rb1 and TP53 and CDK inhibitors p21, p27, p16, and p15 (boxed with Cdkn2a-Rb1) ( Figure 1C)。Enrichment of the expression of dormant genes such as NR2F1, DEC2 (Bhlhe41), TWIST1, CDH5, STAT3, and COL4a5 was also observed in these cells (Kim et al., “Dormancy Signatures and Metastasis in Estrogen Receptor Positive and 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), the entire contents of each of which are incorporated herein by reference) (box containing Nr2f1 - Ccnd1). Another group of DCCs, Group 2 (22%), also showed high levels of expression of ER stress genes together with p21. Group 3 (6%) showed less ER stress, cell cycle inhibitors, and dormant genes, indicating that these genes may represent cells migrating out of dormancy or in a slow - cycling mode. Overall, approximately 40% of DCCs showed high to moderate levels of ER stress gene expression concurrently with cell cycle inhibitors or dormant genes. This is within the percentage range of dormant DCCs detected in progressive MMTV - HER2 animals using phosphorylated histone H3 and phosphorylated Rb detection methods (Harper et al., “Mechanism of Early Dissemination and Metastasis in Her2 + Mammary Cancer,” Nature 540:588 - 592 (2016), the entire contents of which are incorporated herein by reference). Overall, this data indicates that even in animals with detectable metastases, approximately 40% of DCCs exhibit high expression of cell cycle inhibitors. Importantly, in this model, this subset of dormant DCCs showed a peculiar UPR with significant activation of PERK pathway genes.

[0203] Example 3 – Inhibition of PERK eliminates quiescent DCCs in the bone marrow and lung, thereby inhibiting lung metastasis

[0204] The results of the above examples prompted an evaluation of the effects of selective PERK inhibitors on the fate of dormant DCCs and the formation of metastases. LY2, LY3, and LY4 (LY series inhibitors) have been identified as potent and selective PERK inhibitors with appropriate 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), the entire content of which is incorporated herein by reference). The LY series inhibitors were tested in an in vitro kinase assay (using eIF2α as a substrate) and cell-based assays, and eIF2α phosphorylation and its downstream output ATF4 were studied (Table 6). All three inhibitors showed similar or higher 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), the entire content of which is incorporated herein by reference); effectively reduced the levels of P-PERK (P-T980) and its downstream target ATF4 in HER2-expressing MCF10A cells ( Figure 2C and Figure 3A ); and sensitized these same cells to low-dose thapsigargin treatment, demonstrating how these PERK inhibitors selectively affect adaptation to ER stress ( Figure 2D ).

[0205] Using a biochemical enzyme assay ( Figure 2E ), LY4 showed the highest specificity, not presenting secondary kinase targets at concentrations below 15 μM, while LY2, LY3, and GSK2656157 presented several secondary targets at concentrations below 5 μM and even at 1 μM. Using DicoveR x scanMAX TMKinase assays (Table 6) confirmed that LY4 had higher selectivity compared to other inhibitors, even at very high concentrations (20 μM), where LY4 inhibited only 20 out of 456 kinases by >50%, compared to 80 kinases inhibited by >50% by GSK2656157; or LY4 inhibited only 8 kinases by >60%, compared to 58 kinases inhibited by >60% by GSK2656157. None of these secondary targets were any of the other known eIF2α kinases, namely EIF2AK1 (also known as HRI), EIF2AK2 (also known as PKR), and EIF2AK4 (also known as GCN2) (Table 7), indicating that the activity measured on eIF2α was highly specific for PERK inhibition.

[0206] Table 6. Enzymatic and cell-based IC 50 values and kinase selectivity

[0207]

[0208] a PERK biochemical assay using purified eIF2a as substrate.

[0209] b Cell-based assay of tunicamycin-induced eIF2a phosphorylation in 293 cells.

[0210] c Cell-based assay of tunicamycin-induced ATF4-Luc activity in 293 cells.

[0211] d GCN2 biochemical assay using purified eIF2a as substrate.

[0212] e DiscoveR x scanMAX TM kinase assay testing 456 kinases.

[0213] f Atkins et al., “Characterization of a Novel PERK Kinase Inhibitor with Antitumor and Antiangiogenic Activity,” Cancer Res. 73(6):1993 - 2002 (2013), the entire content of which is incorporated herein by reference.

[0214] Table 7. In vitro inhibition comparison of other eiF2α kinases

[0215]

[0216] a Discovery based on binding data using active site probe displacement x scanMAX TM Kinase assays were performed testing 456 kinases.

[0217] b Atkins et al., “Characterization of a Novel PERK Kinase Inhibitor with Antitumor and Antiangiogenic Activity,” Cancer Res. 73(6):1993 - 2002 (2013), the entire content of which is incorporated herein by reference.

[0218] Virgin MMTV - HER2 female mice, 24 - 32 weeks of age, were treated with vehicle or LY4 (50 mpk) by daily intraperitoneal injection for two weeks. Mammary glands, lungs, pancreas, bone marrow, and tumors were collected for further analysis. LY4 was well - tolerated with no significant change in 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), the entire content of which is incorporated herein by reference). No effect on total cell counts in MMTV - HER2 females indicates that the inhibitor has no significant effect on bone marrow cell homeostasis or peripheral blood leukocytes ( Figure 2F )

[0219] Inhibition of PERK led to a significant decrease in the levels of P - PERK and P - eIF2α in mammary ducts and pancreatic tissues (although only partially in islets specifically) ( Figure 3B)。The conclusion is that systemic LY4 delivery can effectively inhibit PERK activation and eIF2α phosphorylation. The inhibition of PERK does not completely deplete PERK activity, which may enable the mice to control their 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), the entire content of which is incorporated herein by reference).

[0220] A high percentage of MMTV-HER2 animals develop lung metastases, which can start early in the progression (Guy et al., “Expression of the Neu Protooncogene in the Mammary Epithelium of Transgenic Mice Induces Metastatic Disease,” Proc. Nat’l. Acad. Sci. U.S.A. 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 content of these references is incorporated herein by reference).

[0221] Therefore, the effect of the LY4 PERK inhibitor on metastatic disease was monitored in animals with small and / or significantly large tumors. All vehicle-treated animals exhibited metastases, which could be detected in H&E-stained sections. Lesions showing >100 cells were classified as macro-metastases because they were also typically positive for proliferation markers ( Figure 1A)。Quantification of macrometastases (5 non-consecutive lung sections) per animal showed that after only two weeks of treatment, LY4 decreased the number and incidence of macrometastases ( Figure 3C ) without affecting the area of these metastases ( Figure 4A ). This suggests that inhibition of PERK may act in the initial stages of metastasis rather than shrinking established macrometastases. Therefore, it was next evaluated whether treatment with LY4 would affect the infiltration of tumor cells from the primary site or the transition from solitary DCCs to micrometastases (containing 2 - 100 cells). Detection of HER2 + circulating tumor cells (CTCs) in blood samples directly showed no significant difference between vehicle- and LY4-treated animals ( Figure 4B ), indicating that LY4 did not severely affect tumor cell infiltration. On the other hand, detection of micrometastases and solitary DCCs by IHC using HER2 revealed a significant decrease in the number of micrometastases in LY4-treated females ( Figure 3D ). More than 80% of solitary DCCs in the lung were negative for P-Rb, indicating that most of them were non-circulating and in a dormant state. This measurement replicated the measurement of a previous study (Harper et al., “Mechanism of Early Dissemination and Metastasisin Her2 + Mammary Cancer,” Nature 540:588 - 592 (2016), the entire content of which is incorporated herein by reference). Significantly, LY4 greatly reduced the number of non-proliferating (P-Rb-negative) solitary DCCs that are normally associated with pulmonary blood vessels without affecting the number of solitary DTCs that are P-Rb positive ( Figure 3E ) or micrometastases ( Figure 4C ). Importantly, LY4 significantly reduced the number of DCCs found in the bone marrow ( Figure 3F)。In this organ, metastasis never occurs, but the incidence of DCC is high and it is in a dormant state (Bragado et al., “TGF-Beta2 Dictates Disseminated Tumour Cell Fate in Target Organs Through TGF-Beta-RIII and P38Alpha / Beta Signalling,” 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 + Mammary Cancer,” Nature 540:588-592 (2016), the entire contents of each of these references are incorporated herein by reference). These results suggest that inhibition of PERK selectively targets non-proliferative dormant DCCs that exhibit active PERK and UPR signalling.

[0222] To further test whether LY4 treatment can selectively target the survival of human DCCs outside the cell cycle, this biology was modelled in 3D cultures. Human ZR75.1 HER2 stably expressing a photoconvertible fluorescent protein (Dendra2) fused to histone H2B +Cells were used for long-term label retention assays because H2B-containing nucleosomes turn over slowly in quiescent cells (Wilson et al., “Hematopoietic Stem Cells Reversibly Switch From Dormancy to Self-Renewal During Homeostasis and Repair,” Cell 135:1118-1129 (2008), the entire content of which is incorporated herein by reference). After exposure to light at 405 nm for approximately 1 minute, the H2B-DENDRA2 protein changes from green to red fluorescence, becoming double positive for green and red; cells that revert to green have divided and diluted the H2B-DENDRA2-RED molecules, 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), the entire content of which is incorporated herein by reference). Cells seeded at high density (clusters) in 3D Matrigel matrix to mimic macrometastases or at low density (single cells) in 3D Matrigel matrix to mimic single DCCs were photoactivated (100%) ( Figure 4D ). After eight days, only 35% of the high-density cells were H2B-DENDRA2-RED positive. In contrast, 65% of the low-density ZR75.1 HER2 + cells were H2B-DENDRA2-RED positive ( Figure 4E ), mimicking the quiescent state of isolated DCCs (Bragado et al., “TGF-Beta2 Dictates Disseminated Tumour Cell Fate in Target Organs Through TGF-Beta-RIII and P38Alpha / Beta Signalling,” Nat. Cell Biol. 15:1351-1361 (2013), the entire content of which is incorporated herein by reference). Treatment with the PERK inhibitor LY4 had no significant effect on the viability of ZR75.1 HER2 + seeded at high density ( Figure 4F ). However, it eliminated quiescent single ZR75.1 HER2 + cells, which is consistent with the in vivo DCC results ( Figure 3G)。These data indicate that the intrinsic and / or ECM-dependent signals in the environment of isolated tumor cells trigger a dependence on PERK signaling, and that LY4 does selectively target slow-cycling or non-proliferative DCC, which is then reactivated to give rise to metastasis.

[0223] Example 4 – PERK inhibition blocks HER2-driven early and late stage mammary tumor progression

[0224] The quiescent UPR, which has been shown to be most associated with dormancy 高 DCC is dependent on PERK, and tumor lesions were next evaluated. In the MMTV-HER2 model, HER2-driven progression was found to be genetically dependent on the PERK kinase (Bobrovnikova-Marjon et al., “PERK-Dependent Regulation of Lipogenesis During Mouse Mammary Gland Development and Adipocyte Differentiation,” Proc. Nat’l. Acad. Sci. U.S.A. 105:16314-16319 (2008), the entire content of which is incorporated herein by reference), and HER2 + tumors have been shown to be sensitive to proteotoxicity and dependent on ERAD (Singh et al., “HER2-mTOR Signaling-Driven Breast Cancer Cells Require ER-Associated Degradation to Survive,” Sci. Signal. 8:ra52 (2015), the entire content of which is incorporated herein by reference). Additionally, 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), the entire content of which is incorporated herein by reference) analysis showed that approximately 14% of HER2-amplified human mammary tumors showed upregulation of the mRNA of PERK ( Figure 5A)。Therefore, it was investigated whether LY4 affects HER2-induced mammary tumor progression in primary lesions, in which the different stages of progression from hyperplastic mammary glands to DCIS and invasive carcinoma can be dissected (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 these references are incorporated herein by reference).

[0225] Analysis of virgin female mammary glands at 24 weeks of age showed that vehicle-treated MMTV-HER2 animals exhibited ducts with secondary and tertiary dense branching ( Figure 6A , left panel), and histological analysis revealed frequent hyperplastic mammary lesions ( Figure 6A , right panel, black arrows). In contrast, LY4-treated animals showed “normalized” glandular architecture with less dense branching, similar to the mammary tree of non-transgenic normal FVB mice ( Figure 3B ). LY4-treated animals also showed a dramatic increase in the number of hollow mammary ducts, constituting more than 60% of the structure, compared to 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 that of vehicle-treated animals. Evaluation by uneven levels of cytokeratin 8 / 18 expression revealed that hyperplastic lesions in control HER2 + animals showed varying degrees of luminal differentiation ( Figure 6C, myoepithelial cells (detected by smooth muscle actin SMA, positive) that are evenly distributed in normal FVB animal ducts are unevenly distributed in the vehicle-treated hyperplasia in MMTV-HER2 mice. In contrast, LY4-treated MMTV-HER2 animals showed increased expression of cytokeratin 8 / 18 in the luminal layer (which usually surrounds the empty duct lumen) and the outer continuous layer of myoepithelial cells ( Figure 6C , lower panels and graphs). This data indicates that LY4 treatment leads to the "normalization" of early cancer lesions through a mechanism that appears to restore the differentiation program.

[0226] Once the animals showed tumors in the size range of 30 to 200 mm 3 (two tumors were >200 mm 3 ), the animals were treated with LY4 for two weeks ( Figure 7A ). In the vehicle-treated group, the tumors grew steadily ( Figure 8A ) and reached 10 times their original volume within two weeks ( Figure 7B , upper panel). In contrast, LY4-treated tumors showed a reduced growth rate ( Figure 8A ), with some tumors remaining in complete cell stasis (defined as the tumor volume doubling only once within two weeks, 43% in the LY4-treated group compared to 7% in the control) ( Figure 7B ) and some tumors (25%) showing regression during the 2-week window of treatment ( Figure 7C ). This led to a significant reduction in the median final tumor volume ( Figure 8B ). Although there was no difference in the proliferation level (P-histone H3 IHC) between vehicle- and LY4-treated tumors ( Figure 7D ), TUNEL staining of tumor sections showed a significant increase in the level of DNA fragmentation in LY4-treated animals ( Figure 8C ). Thus, in the primary lesions, LY4 treatment induced apoptosis in established HER2 + tumors, confirming the dependence on adaptive promotion of PERK function during progression.

[0227] Treatment of 3D acinar cultures of human cancer cells with HER2 overexpression (MCF10A-HER2 or ZR75.1) or HER2 amplification (SKBR3) ( Figure 8D and Figure 7E ) with LY4 in Matrigel showed that treatment with vehicle or LY4 (2 μM) for 10 days significantly increased the level of apoptosis (cleaved caspase-3) in these organoids, especially in the inner cell mass deprived of contact with the ECM ( Figure 8D). Similar to in vivo, no significant changes in proliferation levels detected by phosphorylated histone H3 levels were observed ( Figure 7F ). The conclusion is that early MMTV-HER2 + lesions require PERK to cause HER2-driven ductal epithelial tissue changes. In human cancer cells and mouse tumors with HER2 + , the survival of HER2 depends on PERK.

[0228] Example 5 – Optimal HER2 phosphorylation, localization, and activation of AKT and ERK require PERK signaling

[0229] Since HER2 + 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), the entire content of which is incorporated herein by reference), PERK inhibitors were evaluated to determine whether they would affect optimal HER2 activity due to increased ER client protein load. Detection of HER2 phosphorylation at residues Y1221 / 1222 in tumors showed that the P-HER2-positive regions reported by others (DiGiovanna et al., “Active Signaling by Neu in Transgenic Mice,” Oncogene 17:1877-1884 (1998), the entire content of which is incorporated herein by reference) overlapped with staining for P-PERK and P-eIF2α ( Figure 9A ). This finding indicates that activation of the PERK and HER2 pathways is co-localized. Similarly, single-cell targeted gene expression analysis of primary tumor cells also showed a population of primary tumor cells (approximately 25%) with high levels of ER stress gene expression ( Figure 9B ), which may correspond to primary tumor cells showing P-HER2 activation. Importantly, when scoring P-HER2 levels in tumors, when considering both the area and intensity of staining ( Figure 10A ), it was found that tumors treated with LY4 showed significantly lower levels of P-HER2 than control animals ( Figure 9C)。HER2 signals conduct by forming homodimers or heterodimers 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), the entire contents of which are incorporated herein by reference). In vitro treatment of starved MCF10A-HER2 cells treated with EGF (100 ng / ml, 15 minutes) in the presence or absence of LY4 (2 μM) showed that the PERK inhibitor simultaneously reduced both basal and EGF-induced levels of P-EGFR and P-HER2, while the levels of the survival pathways P-AKT, P-S6 and P-ERK1 / 2 were downregulated ( Figure 9D and Figures and Figure 10B)。Determined by surface biotinylation and co-immunoprecipitation studies, no significant effect on total HER2 levels or heterodimerization with EGFR was observed under these conditions. Since LY4 has no direct inhibitory effect on the active sites of either HER family member AKT or S6 kinase (Table 8), this effect should be attributed to the indirect effect of PERK inhibition on HER2 signaling. In contrast to other HER family members, HER2 is known to remain on the plasma membrane 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), the entire contents of each of the cited documents are incorporated herein by reference). To test whether LY4 would interfere with the activation mechanism of the HER2 receptor, a surface biotinylation assay was performed to measure the presence of the receptor on the cell surface, and receptor endocytosis was determined by reversible surface biotinylation (Cihil et al., “The Cell-Based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins,” J.Vis.Exp.e50867(2013), the entire contents of which are incorporated herein by reference). The data showed that treatment with LY4 reduced the amounts of P-HER2 and total HER2 in the cell surface ( Figure 9E and Figure 10C ), accompanied by an increase in endocytosed phosphorylated HER2 and total HER2 ( Figure 9F ). This data, along with Singh et al., “HER2-mTOR Signaling-Driven Breast Cancer Cells Require ER-Associated Degradation to Survive,” Sci.Signal. 8:ra52(2015) (the entire contents of which are incorporated herein by reference) indicates that PERK signaling and proper UPR function are required to maintain proper HER2 downstream signaling by affecting optimal receptor localization and activation.

[0230] Table 8. Direct inhibitory activity of LY4 on HER family signaling pathways

[0231]

[0232] 1 Discovery using active site probe displacement based on binding data x scanMAX TM Kinase assays were performed on 456 kinases.

[0233] Example 6 - Sequential combination of CDK inhibitor and PERK inhibition enhances the anti - metastatic effect of LY4

[0234] The effect of pharmacological inhibition of PERK on the primary lesion was established and, most importantly, it was also determined that LY4 can inhibit metastasis by eradicating dormant DCC. With this information, it was possible to evaluate whether clinically available drugs capable of mimicking dormancy could be used to render dormancy - induced cancer cells UPR 高 and sensitive to LY4. This hypothesis was supported by the following finding: UPR 高 expresses higher levels of CDK inhibitors in DCC( Figure 1C ). Therefore, it was next investigated whether boosting the quiescent DCC pool by more than 50% above baseline ( Figure 11A ) by pre - treating animals with the CDK4 / 6 inhibitor abemaciclib (50 mpk, 4 weeks) would further enhance the anti - metastatic effect of LY4. Indeed, pre - treating MMTV - HER2 females with abemaciclib alone led to a significant increase in GADD34 + cells in primary tumor sections( Figure 11B ), which otherwise showed very low and focal levels of GADD34 staining (control). Measurements in the primary tumor can be used as a surrogate biomarker for UPR associated with quiescence induced by abemaciclib. As expected, treatment with LY4 eliminated the expression of GADD34 in the primary tumors of treated animals( Figure 11B ). Sequential treatment of mice with abemaciclib (dormancy - like induction phase) and LY4 (dormant DCC eradication phase) led to the same reduction in macrometastatic burden as that observed with single treatment with LY4( Figure 11C ). However, the combination almost completely eliminated the presence of micrometastases( Figure 11D ), and, as seen with single agents, it greatly reduced the number of quiescent single - disseminated cancer cells( Figure 11E ). In summary, these results support that sequential combination of a cell growth inhibitor (such as a CDK inhibitor) and LY4 is a promising therapeutic strategy for preventing metastasis by targeting quiescent DCC that can re - activate and seed these lesions.

[0235] Example 7 - Discussion of Examples 2 - 6

[0236] In HER2 + Multiple studies in breast cancer models have concluded that HER2 +The tumorigenesis of breast cancer 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 have also been found to exist in the surgical margins and in the form of dormant disseminated cancer cells in target organs (Bragado et al., “TGF-Beta2 Dictates Disseminated Tumour Cell Fate in Target Organs Through TGF-Beta-RIII and P38Alpha / Beta Signalling,” Nat. Cell. Biol.15:1351-1361(2013); Chéry et al., “Characterization of Single Disseminated Prostate Cancer CellsReveals Tumor Cell Heterogeneity and Identifies Dormancy AssociatedPathways,” Oncotarget 5(20):9939-51(2014); Sosa et al., “Mechanisms of DisseminatedCancer Cell Dormancy: An Awakening Field,” Nat. Rev. Cancer 14:611-622(2014); andSosa et al., “NR2F1 Controls Tumour Cell Dormancy Via SOX9-and RARbeta-DrivenQuiescence Programmes,” Nat. Commun. 6:6170(2015), each of which is incorporated herein by reference in its entirety), activated the PERK signaling, thereby surviving along with other ER stress pathways (Adomako et al., “Identification of Markers that Functionally Define a Quiescent MultipleMyeloma Cell Sub-Population Surviving Bortezomib Treatment,” BMC Cancer 15:444(2015); Ranganathan et al., “Dual Function of Pancreatic Endoplasmic ReticulumKinase in Tumor Cell Growth Arrest and Survival,” Cancer Res. 68:3260-3268(2008); Ranganathan et al., “Functional Coupling of p38-Induced Up-Regulation of BiPand Activation of RNA-Dependent Protein Kinase-Like Endoplasmic ReticulumKinase 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. U.S.A. 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 Chéry 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)(the entire contents of which are incorporated herein by reference) validated this work by showing that pancreatic DCCs present in the liver (and other models) activate the UPR at rest.

[0237] Examples of the present disclosure confirm that the PERK inhibitor LY4 can selectively target DCC and HER2-dependence in the primary tumor. An important finding to be discussed is the inhibitory effect of LY4 on metastasis. As in patients, metastasis in the MMTV-HER2 model may be asynchronous with the primary tumor and sometimes even develop occult primary lesions, where some metastases start before the detection of an obvious tumor (Husemann et al., “Systemic Spread Is an Early Step in BreastCancer,”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 EarlyDissemination 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), the disclosures of which are incorporated herein by reference in their entireties). LY4 treatment reduced all metastases: early metastases (before the confirmed tumor was palpable) or metastases that occurred concurrently with the growth of the confirmed primary tumor (Figure 8). This is important because it indicates that the effect on metastases is not solely due to LY4 reducing the primary tumor burden. Unexpectedly, LY4 treatment reduced the metastatic burden by eliminating non-proliferating single or small clusters of P-Rb negative DTCs. Imaging and single-cell multiplex qPCR revealed that these DCCs showed more frequent upregulation of GADD34 (protein) and a large number of ER stress genes (including PERK itself), and also exhibited a quiescent phenotype, as revealed by the upregulation of several negative regulators of cell proliferation. It should be considered that part of the PERK-induced ER stress program requires transcriptional regulation, while another part is the preferential translation of upstream ORF-containing genes 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), the entire content of which is incorporated herein by reference). Similarly, it has been shown that UPR-induced G1 arrest is caused by inhibiting the translation of cyclin D1 (Brewer et al., “Mammalian Unfolded Protein Response Inhibits Cyclin D1 Translation and Cell-Cycle Progression,” Proc. Natl. Acad. Sci. 96:8505-8510 (1999), the entire content of which is incorporated herein by reference). Using human HER2 +3D organogenesis experiments of cancer cell lines confirmed the selective killing of LY4 on quiescent single cancer cells. These data indicate that quiescent DCCs are more likely to rely on PERK signaling for survival. Similarly, a subset of human metastatic cells from breast cancer patients also showed a negative correlation between GADD34 and Ki67, validating the correlation found in mice. These data suggest that, together with NR2F1 (Borgen et al., “NR2F1 Stratifies Dormant Disseminated Tumor Cells in Breast Cancer Patients,” Breast Cancer Research 20:120 (2018), the entire content of which is incorporated herein by reference), GADD34 can serve as a reliable biomarker for dormant / UPR-high DCCs when used alone or in combination with NR2F1, thus providing guidance for selecting patients for treatment.

[0238] It is important to identify targets and drugs that can eliminate quiescent DCCs, as quiescent DCCs are known to evade anti-proliferative therapies through both 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 Tumour Cells is Preset by Primary Tumour 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, where these cells are also typically in a dormant state, (Bragado et al., “TGF-Beta2 Dictates Disseminated Tumour Cell Fate in Target Organs Through TGF-Beta-RIII and P38Alpha / Beta Signalling,” Nat. Cell Biol. 15:1351-1361 (2013); Chéry 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 Tumour 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), each of which is incorporated herein by reference in its entirety), further emphasizes the concept that PERK inhibition is a therapy for dormant DCCs (Aguirre-Ghiso et al., “Metastasis Awakening: Targeting Dormant Cancer,” Nat. Med. 19:276-277 (2013), the entire content of which is incorporated herein by reference), which can be used in an adjuvant setting to eliminate minimal residual dormant disease (Aguirre-Ghiso et al., “Metastasis Awakening: Targeting Dormant Cancer,” Nat. Med. 19:276-277 (2013), the entire content of which is incorporated herein by reference).

[0239] The exact mechanism by which PERK kinase inhibition blocks tumor growth is not yet clear. A reduced 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), the entire content of which is incorporated herein by reference) may be one mechanism. The results described herein demonstrate that LY4 reduces the level of phosphorylated HER2 in vivo, and that LY4 reduces the abundance of active receptors in the membrane through enhanced endocytosis. No change in HER2 protein degradation was observed. Regarding how PERK precisely controls the membrane localization or endocytosis of HER2, it is possible that receptor internalization enables better or faster dephosphorylation of the receptor or reduces the chance of receptor activation, resulting in reduced downstream signaling. It has been shown that endocytosis of receptors can reduce the signal output of many plasma membrane-localized receptors 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), the entire content of which is incorporated herein by reference).

[0240] The results herein further demonstrate that in early lesions, LY4 induces a differentiated phenotype. However, in established tumors, when LY4 is used as a single agent, it can push the tumor into a state of stasis or regression. This suggests that in the early stage, HER2 + Dysregulation of PERK signaling in early lesions is more associated with the loss of the differentiation program, but this mechanism remains to be determined. Then, as the biology of the tumor changes to become highly proliferative, these HER2 + tumors remain highly dependent on PERK. This may be related to changes in HER2 function during progression; in the early stage, it mainly deregulates the morphogenetic program, leading to anoikis resistance and dissemination, while in the later stage it is mainly involved in proliferation and survival programs.

[0241] The results described herein also point to the value of combining standard anti-proliferative therapies with LY4, which can eliminate residual quiescent cells. This approach was tested by sequentially using a combination of the CDK4 / 6 inhibitor abemaciclib and LY4, and the results showed an improved anti-metastatic effect. Encouragingly, the LY4 dose used did not significantly affect glucose levels, bone marrow or peripheral blood cell counts, water intake, or feeding behavior in non-tumor or tumor-bearing mice. This indicates that the dose used, while severely impeding tumor growth and metastasis by eradicating dormant DCC, did not affect normal organ function in the host. Since it was also found that dormant / UPR 高 DCC downregulated the surface expression of MHC-1 (Pommier et al., “Unresolved Endoplasmic Reticulum Stress Engenders Immune-Resistant, Latent Pancreatic Cancer Metastases,” Science 360(6394):eaao4908 (2018) (the entire content of which is incorporated herein by reference), LY4 may also help the adaptive immune response target DCC and potentially established tumors. Current work is investigating this possibility. The results described herein open the door to using anti-dormant DCC survival therapies as a new approach to target metastatic disease. This would target the full phenotypic heterogeneity of disseminated disease, which may include proliferative, slow-cycling, and dormant DCC (Aguirre-Ghiso et al., “Metastasis Awakening: Targeting Dormant Cancer,” Nat. Med. 19:276-277 (2013), the entire content of which is incorporated herein by reference).

[0242] Example 8 – Combination of the CDK4 / 6 inhibitor abemaciclib and the PERK inhibitor LY4 in melanoma cell lines

[0243] Since CDK4 / 6 inhibitors have been shown to induce cell cycle arrest and LY4 to induce cell death in dormant cell cycle-arrested DTCs ( Figure 12A ), the combination of a CDK4 / 6 inhibitor and LY4 was then investigated to determine whether it would reduce cell viability in in vitro cell lines. In 2D and 3D in vitro cell cultures, the CDK 4 / 6 inhibitor abemaciclib has been shown to inhibit the growth of WM35 melanoma cells. Pretreatment with 50 nM abemaciclib (2D) for 1 week followed by an in vitro acute treatment (48 hours) with 2 μM LY4 reduced the viability of Braf-mutant melanoma WM35 cells compared to treating the cells with 2 μM LY4 alone ( Figure 12B)。In in vitro 3D cultures, the addition of 2 μM LY4 after 1 week of abemaciclib pretreatment had an additive effect on reducing cell viability ( Figure 12C )。 Figure 12C The results shown indicate that abemaciclib pretreatment may induce growth arrest and some cell death in 3D cell cultures. Subsequently, the addition of LY4 seems to enhance the effect on cell death. This is consistent with the idea that cells arrested by abemaciclib may upregulate the ER stress response, as shown by the upregulation of GADD34 ( Figure 12G ) and are subsequently sensitive to LY4.

[0244] In melanoma cells, an abemaciclib-resistant phenotype emerged after 4 - 5 weeks of continuous treatment. Co-treatment of cells with LY4 and abemaciclib reduced the number of abemaciclib-resistant cells surviving in 2D cell cultures but did not show the enhancement expected from these experiments in 2D cultures. However, in abemaciclib-resistant cells, LY4 had an additive effect on reducing the viability of 3D cell cultures after continuous treatment with abemaciclib ( Figures 12D - 12E )。These data indicate that cells resistant to melanoma CDK4 / 6 inhibitors remain dependent on the PERK-mediated ER stress response for survival. In in vitro 3D cultures, the addition of LY4 after 5 weeks of abemaciclib pretreatment had an additive effect on reducing cell viability ( Figure 12F ), which could be determined by apoptosis of cells taking up DAPI. Although resistant cells grew as well as control cells in 2D cultures, abemaciclib pretreatment seemed to induce cell death in 3D cultures ( Figure 12F )。

[0245] Example 9 – BMP7-F9 Induces and Maintains Dormancy of DTCs (HNSCC)

[0246] BMP7-F9 reduced the ERK / p38 activity ratio and induced various mRNAs in the induction of dormancy characteristics ( Figures 13A - 13C )。 Figure 13A Shown is that treatment with 2 ng / ml, 5 ng / ml, and 10 ng / ml of BMP7-F9 (the second, third, and fourth gray bars respectively; control is the first black bar) reduced the ERK / p38 activity ratio compared to control, as determined by Western blotting in HEp3 HNSCC cells. The effect on the ERK / p38 activity ratio was observed at 2 - 6 and 24 hours (the second to fourth sets of bars). BMP7 stimulated ERK activity within the first 30 minutes (the first set of bars). Figure 13B Shown is that treatment with BMP7-F9 induced DEC2, p53, and p27 mRNAs encoding genes for dormancy characteristics (10 ng / ml BMP7-F9, 24 hours).Figure 13C It was shown that treatment of the same cells with BMP7-F9 (10 ng / ml, 24 hours) by immunofluorescence induced nuclear accumulation of NR2F1, a strong dormancy-inducing transcription factor. Figure 13A and Figure 13B The differences in

[0247] BMP7-F9 induced growth arrest of T-HEp3 cells in vitro and in vivo ( Figures 14A - 14E ). Figure 14A It was shown that treatment of T-HEp3 cells with BMP7-F9 inhibited their in vitro proliferation for 48 hours, as determined by CellTiter-Blue assay (RFU, relative fluorescence unit). Figure 14B is Figures 14C - 14D A schematic diagram of the in vivo experimental procedure used in Figure 14C . T-HEp3 cells were pretreated with BMP7-F9 in vitro for 24 hours and then inoculated on the chorioallantoic membrane (CAM) of chicken embryos ( Figure 14D ), and on said membrane they were treated in vivo daily with vehicle or BMP7-F9 (50 ng / ml), and subsequently tumors were collected and the number of HEp3 HNSCC cells / tumors ( Figure 14E ) and the level of P-H3 (

[0248] NSG mice were treated according to the method in Figure 15A for 3 and 6 weeks. The percentage of local recurrence and DTC occurrence was scored at these time points. The tabulated results corresponding to Figure 15B showed that BMP7 limited the incidence of local recurrence after tumor surgery (Table 9) and the incidence of DTC in the lung (Table 10), and as shown below (Table 11).

[0249] Table 9. Effect of BMP7-F9 on local recurrence at the surgical margin in the adjuvant setting

[0250]

[0251]

[0252] Table 10. Effect of BMP7-F9 on the incidence of DCC in the lung

[0253]

[0254] Table 11. In the same experiment as shown in Figures 15A - 15B and Tables 9 and 10, the median of GFP + / vimentin + tumor cells / lung.

[0255]

[0256] HEp3-GFP HNSCC tumors grew until approximately 300 mm 3 and were then treated with 50 μg / kg BMP7-F9 in a neo-adjuvant setting until the tumors were approximately 600 mm 3 . The tumors were then resected surgically. One to two days after surgery, adjuvant treatment with BMP7-F9 was continued for 4 weeks. The animals were then euthanized and the DCC burden in the lungs was scored using a fluorescence microscope. It was observed that BMP7 restricted the development of local and distant recurrence after tumor surgery. NSG mice were treated according to the method in Figure 15A for 4 weeks. At these time points, the percentage of local recurrence and DCC occurrence was scored. The number of GFP-positive cells in the isolated lungs was scored after treatment. This is a measure of the DCC burden in the lungs, which was significantly reduced by treatment with BMP7-F9. Note that the median DCC burden decreased by one log, and BMP-7 significantly cured DCC in 3 out of 7 animals.

[0257] In the neo-adjuvant + adjuvant setting, the effects of BMP7-F9 on local recurrence at the surgical margin (Table 12) and the incidence of DCC in the lungs (Table 13) are shown below. The results are tabulated from the results in Figure 15C where the treatment of the mice was the same as in Figure 15A except that the adjuvant treatment lasted for 4 weeks. The number of GFP-positive cells in the isolated lungs was scored after treatment. The results showed that treatment with BMP7 in the neo-adjuvant and adjuvant settings after tumor surgery restricted the incidence of local recurrence (Table 12) and the incidence of DCC in the lungs (Table 13). Table 14 shows the measure of the DCC burden in the lungs, which was significantly reduced by treatment with BMP7-F9. Note that the median DCC burden decreased by one log, and BMP-7 significantly cured DCC in 3 out of 7 animals.

[0258] Table 12. Effect of BMP7-F9 on local recurrence at the surgical margin in the neo-adjuvant + adjuvant setting

[0259]

[0260] Table 13. Incidence of DCC in the lungs

[0261]

[0262] Table 14. Median GFP + / vimentin + tumor cells / lung reported in Tables 12 and 13.

[0263]

[0264] Although the preferred embodiments have been described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, etc. can be made without departing from the spirit of the present invention. Therefore, these are considered to be within the scope of the present invention as defined by the appended claims. Sequence Listing <110> Icahn School of Medicine at Mount Sinai <120> Method for Treating Minimal Residual Cancer <130> 147535.00711 (170912) <150> 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 Ala Thr Asn His Ala Xaa Xaa Gln Xaa Leu Xaa His Xaa Xaa Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Xaa Ala Ile 100 105 110 Ser Xaa Leu Tyr Phe Asp Asp Xaa Ser Asn Val Ile Leu Lys Lys Xaa 115 120 125 Arg Asn Met Xaa Val Xaa Ala Cys Gly Cys His 130 135 <210> 4 <211> 139 <212> PRT <213> Homo sapiens <400> 4 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 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. Use of a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor in the preparation of a medicament for treating minimal residual cancer in a subject, wherein the treatment comprises: Contacting disseminated cancer cells (DCC) in a subject with a protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor, wherein the contacting eradicates DCC in the subject to treat minimal residual cancer in the subject; wherein the protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor is 2-amino-5-[4-[[(2R)-2-(3,5-difluorophenyl)-2-hydroxy-acetyl]amino]-2-methyl-phenyl]-N-isopropylpyridine-3-carboxamide, which has the chemical structure shown below: ； and wherein the subject has been diagnosed with human epidermal growth factor receptor 2 positive (HER2 + ) breast cancer.

2. The use according to claim 1, wherein the treatment further comprises: Administering a chemotherapeutic agent, an immunotherapeutic agent, an epigenetic agent, or ionizing radiation to the subject.

3. The use according to claim 2, wherein a chemotherapeutic agent is administered to the subject, and wherein the chemotherapeutic agent is an anti-HER2 chemotherapeutic agent selected from trastuzumab and lapatinib.

4. The use according to claim 2, wherein a chemotherapeutic agent is administered to the subject, and wherein the chemotherapeutic agent is selected from anthracyclines, taxanes, kinase inhibitors, antibodies, fluoropyrimidines, and platinum drugs.

5. The use according to claim 2, wherein an immunotherapeutic agent is administered to the subject, and wherein the immunotherapeutic agent is selected from immune checkpoint inhibitors, interferons, or tumor vaccines.

6. The use according to claim 2, wherein an epigenetic agent is administered to the subject, and wherein the epigenetic agent is selected from histone deacetylase inhibitors, 5-azacytidine, retinoic acid, arsenic trioxide, enhancer inhibitors of the Zeste 2 polycomb repressive complex 2 subunit, or bromodomain inhibitors.

7. The use according to claim 1, wherein the contacting is effected by administering the PERK inhibitor to the subject.

8. The use according to claim 1, wherein the treatment further comprises: Detecting the presence of DCC in the subject prior to the contacting.

9. The use according to claim 8, wherein the DCC is NR2F1 + 。 10. The use according to claim 8, wherein the DCC is active for phosphorylated PERK (phospho-PERK).

11. The use according to claim 8, wherein the DCC is positive for bone morphogenetic protein receptor.

12. The use according to claim 11, wherein the treatment further comprises: Contacting DCC in the subject with a bone morphogenetic protein 7 (BMP7)-derived protein, wherein the BMP7-derived protein is BMP7-F9 having the amino acid sequence shown in SEQ ID NO:

8.

13. Use according to claim 12, wherein contacting DCC with the BMP7-derived protein in the subject is effected by administering the BMP7-derived protein to the subject.

14. Use according to claim 1, wherein the PERK inhibitor does not inhibit EIF2AK1, EIF2AK2 or EIF2AK4.

15. Use according to claim 1, wherein the subject is a human.

16. Use according to any one of claims 1-15, wherein the treatment further comprises: Selecting a subject in remission from cancer prior to the contacting.

Images