Deruption of insulin-like growth factor 2 secretion to treat cancer

Abstract

The present application relates to disruption of insulin-like growth factor 2 (IGF2) secretion to treat cancer, in particular to pharmaceutical compositions and methods for inhibiting IGF2 signaling. IGF2 is a key signaling molecule that plays an important role in a variety of physiological processes, including skeletal muscle generation and adult muscle remodeling during development. Abnormal activation of the signaling pathway induced by IGF2 has been shown to promote cancer progression. A p24 family protein TMED10 plays a role of a cargo receptor, and promotes IGF2 to be output from an endoplasmic reticulum (ER) by recognizing an ER output signal on IGF2. In addition, the TMED10 also mediates the ER output of another sortilin, and the protein is very important for regulating the output of IGF2 from the reverse Golgi body net. The present application features a new therapeutic strategy for the treatment of cancer, including but not limited to inhibitors targeting the interaction between IGF2 and TMED10.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 595,766, filed on November 3, 2023, the entire contents of which are incorporated herein by reference, including any figures, tables, or drawings.

[0003] Description of Sequence Listing

[0004] The name of the sequence listing of this application is "Sequence Listing.txt", which was created on October 28, 2024 and has a length of 12KB. The entire contents of the sequence listing are incorporated herein by reference. Technical Field

[0005] The present application relates to the field of biotechnology, and in particular to a pharmaceutical composition and method for inhibiting IGF2 signal transduction. Background Art

[0006] Insulin-like growth factor 2 (IGF2) is a key player in various cellular processes such as cell proliferation, migration, differentiation and survival. IGF2 is involved in various physiological activities, especially skeletal muscle generation (W. Chao, PAD'Amore, IGF2: epigenetic regulation and role in development and disease. Cytokine Growth Factor Rev 19, 111-120 (2008)). IGF2 is an embryonic regulator of myogenesis and an autocrine factor that promotes the differentiation of myoblasts in vitro (JR Florini, DZ Ewton, SL Roof, Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression. Mol Endocrinol 5, 718-724 (1991); JR Florini et al., "Spontaneous" differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-II. J Biol Chem 266, 15917-15923 (1991); Y. Ge, Y. Sun, J. Chen, IGF-II is regulated by microRNA-125b in skeletal myogenesis. J Cell Biol 192, 69-81 (2011)). Knockdown of IGF2 in mouse muscle myoblasts impaired the differentiation process, demonstrating that IGF2 regulates muscle stem cell differentiation (see JRFlorini above). IGF2 dysregulation has been associated with a variety of pathologies, including Silver-Russell syndrome and Beckwith-Wiedemann syndrome. Defective IGF2 signaling pathways or overexpression of IGF2 trigger uncontrolled cell growth and lead to cancer growth. IGF2 is considered a potential biomarker for different stages of tumor progression and plays a key role in cancer development due to its abnormal expression in tumor tissues. However, despite extensive studies on the expression of IGF2 and the signal transduction pathways it induces, little is known about the mechanisms that control the secretion of newly synthesized IGF2 from producer cells.

[0007] IGF2 works by binding to specific cell surface receptors, thereby starting the downstream signal transduction pathway. In order to inhibit IGF2 signal transduction, strategies can focus on the target receptors necessary for the process, such as the IGF1 receptor (IGF1R), which plays a key role in transmitting IGF2 signals. Preclinical treatment methods using IGF1R inhibitors have been shown to have anti-tumor effects. Alternatively, IGF2 signal transduction can be limited by blocking the secretion of newly synthesized IGF2 protein. Therefore, an effective method for targeting the IGF2 signal transduction pathway is needed. Summary of the invention

[0008] In one aspect, the present application provides a pharmaceutical composition for inhibiting IGF2 signaling, comprising an inhibitor of IGF2-TMED10 interaction in a cell, wherein the cell is a cell of a subject having abnormal IGF2 signaling.

[0009] In another aspect, the present application provides a method for treating a subject with abnormal IGF2 signaling, wherein the method comprises: (a) providing a pharmaceutical composition comprising an inhibitor of IGF2-TMED10 interaction in a cell and a pharmaceutical carrier or excipient; and (b) administering an effective amount of the pharmaceutical composition to the subject.

[0010] In another aspect, the present application provides a method for treating cancer in a subject in need thereof, the method comprising: (a) providing a pharmaceutical composition comprising an inhibitor of IGF2-TMED10 interaction in a cell and a pharmaceutical carrier or excipient; and (b) administering an effective amount of the pharmaceutical composition to the subject.

[0011] In preferred embodiments, the inhibitors include: a human IGF2 polypeptide comprising residues 112-140 of the IGF2 sequence; a human TMED10 polypeptide comprising residues 1-130 of the human TMED10 sequence; a small molecule; a covalent inhibitor or antibody; and combinations thereof.

[0012] In certain embodiments, the subject suffers from Beckwith-Wiedemann syndrome, Russell-Silver syndrome, or Doege-Potter syndrome. In certain embodiments, the subject suffers from cancer. In certain embodiments, the subject is a mammal, which may be a human.

[0013] In certain embodiments, genome editing tools can be used to inhibit the interaction between IGF2 and TMED10 in cells. In certain embodiments, genome editing tools include but are not limited to CRISPR / CAS9 (RNA-guided targeting) genome editing tools, wherein CRISPR / CAS9 genome editing tools are used to mutate the IGF2 sequence encoding residues 112-140 of IGF2, or mutate the human TMED10 sequence encoding residues 1-130 of the human TMED10 sequence, wherein the mutated TMED10 blocks TMED10 from binding to IGF2.

[0014] In preferred embodiments, the inhibitor blocks the secretion of IGF2 in the cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1A to FIG. 1I It was confirmed that knockdown of TMED10 caused a defect in IGF2 secretion. Figure 1A A schematic diagram illustrating the design of the RUSH-IGF2-HA construct and the RUSH assay is shown. Figure 1B HeLa cells transfected with plasmids encoding Str-KDEL and full-length RUSH-IGF2-HA are shown. On day 1 post-transfection, cells were pre-incubated with cycloheximide for 2 hours. Then, after incubation with biotin and cycloheximide for the indicated times, the localization of RUSH-IGF2-HA was analyzed. Views of the indicated areas in panel E are shown in panel E' at higher exposures. Figure 1C Shown are HeLa cells transfected with control siRNA or siRNA against TMED10. Two days after transfection, the levels of TMED10 and β-actin in cell lysates were analyzed by immunoblotting. Figure 1D Cells retransfected with plasmids encoding Str-KDEL and RUSH-IGF2-HA 24 hours after siRNA transfection are shown. On the 3rd day after knockdown, cells were pre-incubated with cycloheximide for 2 hours. Then, cells were incubated with biotin and cycloheximide for 2 hours. After biotin incubation, the level of RUSH-IGF2-HA in culture medium and cell lysate was analyzed by immunoblotting. Figure 1E Shown are quantification results of the abundance of secreted IGF2, normalized to that detected in the cell lysate set in the absence of biotin (mean ± SD; n = 3). Figure 1FCells re-transfected with plasmids encoding Str-KDEL and RUSH-ShhN-HA 24 hours after siRNA transfection are shown. On day 3 after knockdown, cells were pre-incubated with cycloheximide for 2 hours. Then, cells were incubated with biotin and cycloheximide for 2 hours. After biotin incubation, the levels of RUSH-ShhN-HA in the culture medium and cell lysates were analyzed by immunoblotting. Figure 1G Shown are quantification results of the abundance of secreted ShhN, normalized to that detected in the cell lysate set in the absence of biotin (mean ± SD; n = 3). Figure 1H It is shown that on the first day after transfection with siRNA, cells were transiently transfected with RUSH-HA-IGF2. On the third day after knockdown, cells were pre-incubated with cycloheximide for 2 hours. Then, cells were incubated with biotin and cycloheximide for 2 hours. After biotin incubation, the abundance of RUSH-HA-IGF2 in culture medium and cell lysates was analyzed by immunoblotting. Fig. 1I The results of quantitative analysis of the abundance of secreted IGF2 are shown, normalized to the abundance detected in cell lysates in the absence of biotin (mean ± SD; n = 3). In each experimental group of each replicate experiment, the values ​​were normalized to the mean of the blank control group (Mock group) and the TMED10 KD group (H, J, L). ***, p < 0.001; ****, p < 0.0001; ns, no significant difference.

[0016] FIG. 2A to FIG. 2I It was confirmed that TMED10 mediated the release of IGF2 into COPII vesicles. Figure 2A HeLa cells transfected with control siRNA or siRNA against TMED10 are shown. On day 1 after transfection, cells were re-transfected with plasmids encoding Str-KDEL and RUSH-IGF2-HA. On day 3 after knockdown, cells were pre-incubated with cycloheximide for 2 h. Then, cells were incubated with biotin and cycloheximide for 20 min and analyzed for localization of IGF2. Scale bar, 10 μm. Figure 2B Also shown are the abundance of TMED10 and β-actin in cell lysates before biotin treatment analyzed by immunoblotting. A representative example of three biological replicates is shown in this figure. Figure 2C Quantification of the percentage of cells showing juxtanuclear localization of RUSH-IGF2-HA at the indicated time points after biotin treatment is shown (mean ± SD; n = 3; > 100 cells were counted in each experiment). Figure 2D A schematic diagram illustrating the vesicle formation assay is shown. Figure 2E , Figure 2F and Figure 2HShown is the use of HEK293T cells ( Figure 2E ) or HEK293T cells transfected with control siRNA or siRNA targeting TMED10 ( Figure 2F and Figure 2H Vesicle formation assay was performed by immunoblotting. Figure 2G and Fig.2I Quantification of budding efficiency of the indicated proteins from the vesicle formation assay is shown (mean ± SD; n = 3). The budding efficiency was quantified by calculating the abundance of RUSH-IGF2-HA in the vesicle fraction, normalized to the cargo protein level at 1% loading. The value was then normalized to the mean of all experimental groups in each replicate. **, p < 0.01; ***, p < 0.001.

[0017] FIG. 3A to FIG. 3D It was shown that residues 112-140 in IGF2 are required for ER-to-Golgi trafficking of IGF2. Figure 3A Shown is a sequence alignment of IGF2 from different species (SEQ ID NOs: 6-11). Figure 3B HeLa cells transfected with plasmids encoding the indicated RUSH constructs were incubated with biotin and cycloheximide for the indicated time periods. Localization of the indicated proteins was analyzed by immunofluorescence. Scale bar, 10 μm. Figure 3C and Figure 3D Quantitative percentage of cells showing juxtanuclear localization of RUSH constructs after biotin treatment is shown (mean ± SD; n = 3; > 100 cells were counted in each experiment). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significantly different.

[0018] 4A to 4H Residues 112-140 in IGF2 are shown to directly interact with the GOLD domain of TMED10. Figure 4A , Figure 4C and Figure 4E HEK293T cells co-transfected with plasmids encoding the indicated constructs are shown. On day 1 post-transfection, cells were treated with 2 mM DSP and cell lysates were incubated with beads conjugated with anti-FLAG antibodies. Bound proteins were analyzed by immunoblotting. Figure 4B , Figure 4D and Figure 4FQuantification of the relative levels of the indicated proteins immunoprecipitated with FLAG-tagged proteins is shown (mean ± SD; n = 3). Quantification was performed by calculating the abundance of bound proteins normalized to the abundance of the protein in the loading. The values ​​were then normalized to the mean of all experimental groups in each biological replicate. Figure 4G Shown is a peptide corresponding to residues 112-140 of IGF2 covalently linked to thiopyridone Sepharose 6B, incubated with purified GST or GST-tagged human TMED10 GOLD domain (residues 1-130). After incubation, bound proteins were analyzed by immunoblotting. Figure 4H GST-TMED binding to quantified IGF2 peptide is shown. 101-130 The levels of GST and GST-TMED10(1-130) bound to IGF2 peptide were normalized to the average level in each biological replicate. *, p<0.05; **, p<0.01; ****, p<0.0001.

[0019] FIG. 5A to FIG. 5D It was shown that TMED10 plays an important role in the secretion of IGF2 by C2C12 cells for muscle stem cell differentiation. Figure 5A Shown are C2C12 cells transfected with control siRNA or siRNA against TMED10, incubated with differentiation medium (DM) with or without 100 ng / mL purified IGF2. After 3 days of incubation, the expression of the indicated proteins was analyzed by immunoblotting. Figure 5B Relative levels of the indicated proteins were quantified after cell differentiation assays are shown (mean ± SD; n = 3). Quantification results were normalized to the mean level of myogenin in all three experimental groups in each biological replicate. Figure 5C Shown are C2C12 cells transfected with control siRNA or siRNA against TMED10 and incubated for 3 days with DM containing or not containing 100 ng / mL purified IGF2. The morphology of MHC-labeled myotubes was analyzed by immunofluorescence. Scale bar, 200 μm. Figure 5D Quantification of the differentiation index in each designated experimental group is shown (mean ± SD; n = 3). The differentiation index was quantified by calculating the percentage of the number of nuclei in myosin heavy chain (MHC) positive cells relative to the total number of nuclei after the C2C12 differentiation assay. ***, p < 0.001; **, p < 0.01.

[0020] FIG. 6A to FIG. 6G TMED10 is shown to regulate ER-to-Golgi translocation of sortilin (neurotensin receptor-3). Fig. 6A Table showing the list of transmembrane proteins less frequently present in vesicle fractions in the TMED10KO group compared to the WT group (average fold change < 0.5). Figure 6B Vesicle formation assays using HeLa WT or HeLa TMED10 KO cells are shown. The indicated proteins were analyzed by immunoblotting. Figure 6C WT or TMED10 KO HeLa cells transfected with SBP-EGFP-sortilin and Str-KDEL in the presence or absence of TMED10-FLAG are shown. 24 hours after transfection, cells were pre-incubated with cycloheximide for 2 hours. Then, cells were incubated with biotin and cycloheximide for the indicated time points. The localization of the indicated proteins was then analyzed by immunofluorescence. Scale bar, 10 μm. Fig.6D Quantification of the percentage of cells showing juxtanuclear localization of sortilin is shown (mean ± SD; n = 3; > 100 cells were counted for each experiment). ***, p < 0.001; ****, p < 0.0001; ns, not significantly different. Fig. 6E A schematic diagram demonstrating the vesicle immunoprecipitation assay is shown. Fig. 6F and Figure 6G Vesicle formation assays performed in untransfected HeLa cells or in cells co-transfected with plasmids encoding TMED10-HA and RUSH-sortilin-Myc are shown. Vesicles enriched with TMED10-HA were then immunoisolated and analyzed by immunoblotting for the abundance of the indicated proteins in immunoisolated vesicles and in vesicles that were not immunoisolated (flow-through). Fig. 6F and Figure 6G The data shown are representative examples of three biological replicates.

[0021] FIG. 7A to FIG. 7G Shown that sortilin regulates the TGN export of IGF2. Fig. 7A HeLa cells transfected with control siRNA or siRNA against sortilin are shown. Two days after transfection, the levels of the indicated proteins in cell lysates were analyzed by immunoblotting. Figure 7B HeLa cells transfected with control siRNA or siRNA against sortilin are shown. 24 hours after transfection, cells were re-transfected with plasmids encoding Str-KDEL and RUSH-HA-IGF2. On the 3rd day after knockdown, cells were pre-incubated with cycloheximide for 2 hours. Then, cells were incubated with biotin and cycloheximide for 2 hours. After biotin incubation, the levels of RUSH-HA-IGF2 in culture medium and cell lysates were analyzed by immunoblotting. Figure 7CQuantification results of the abundance of secreted IGF2 are shown, normalized to that detected in the cell lysate group (mean ± SD; n = 3). The values ​​in each experimental group were normalized to the mean of the blank control group and sortilin KD group in each biological replicate. Fig.7D HeLa cells transfected with control siRNA or siRNA against sortilin are shown. 24 hours after transfection, cells were re-transfected with plasmids encoding Str-KDEL and RUSH-HA-IGF2. On day 3 after knockdown, cells were pre-incubated with cycloheximide for 2 hours. Then, cells were incubated with cycloheximide and biotin for the indicated times and analyzed for the localization of RUSH-HA-IGF2. Scale bar, 10 μm. Magnified views of the indicated areas in Figures F and I are shown in Figures F', F", I', I". Fig. 7E Quantification of the percentage of cells showing juxtanuclear localization of RUSH-HA-IGF2 is shown (mean ± SD; n = 3; >100 cells were counted in each experimental group). Figure 7F Quantification results of the percentage of cells showing punctate forms of RUSH-HA-IGF2 are shown (mean ± SD; n = 3; >100 cells were counted in each experimental group). Figure 7G A proposed model is shown that illustrates the dual function of TMED10 in mediating IGF2 trafficking along the secretory pathway: (1) the GOLD domain of TMED10 recognizes residues 112-140 of IGF2 to recruit IGF2 to COPII vesicles; (2) TMED10 also regulates the ER export of newly synthesized sortilin, which is important for the TGN-to-plasma membrane trafficking of IGF2. ***, p<0.001; ns, not significantly different.

[0022] FIG. 8A to FIG. 8D It was shown that knockout of TMED10 caused a defect in IGF2 secretion. Fig. 8A Shown are cell lysates of WT or TMED10KO HeLa cells analyzed by immunoblotting using the indicated antibodies. Figure 8B WT or TMED10 KO HeLa cells transiently transfected with RUSH-IGF2-HA are shown. On day 1 after transfection, cells were pre-incubated with cycloheximide for 2 hours. Then, cells were incubated with biotin and cycloheximide for 2 hours. After biotin incubation, the levels of RUSH-IGF2-HA in the culture medium and cell lysates were analyzed by immunoblotting. Figure 8CQuantification of the abundance of secreted IGF2 is shown, normalized to that detected in the cell lysate group (mean ± SD; n = 3). In each replicate experiment, the values ​​in each experimental group were normalized to the mean of the WT and TMED10 KO groups. **, p < 0.01. Fig.8D TMED10 KO HeLa cells transiently transfected with RUSH-HA-IGF2 are shown. On day 1 post-transfection, cells were pre-incubated with cycloheximide with / without 100 nM bafilomycin A1 or 50 μM MG132 for 2 hours. Cells were incubated with biotin for 2 hours. After incubation, the levels of RUSH-IGF2-HA in cell lysates were analyzed by immunoblotting.

[0023] 9A to 9D It was shown that knockout of TMED10 caused a defect in ER-to-Golgi trafficking of IGF2. Fig. 9A and Fig. 9C WT or TMED10 KO HeLa cells transiently transfected with plasmids encoding RUSH-IGF2-HA (AR and TY) or co-transfected with plasmids encoding RUSH-IGF2-HA and TMED10-FLAG (Z-AB) are shown. On day 1 after transfection, cells were pre-incubated with cycloheximide for 2 h. Then, cells were incubated with biotin and cycloheximide at 37°C for the indicated time points, and the localization of RUSH-IGF2-HA was analyzed by immunofluorescence. Scale bar, 10 μm. Fig. 9B and Fig.9D Quantification of the percentage of cells showing the juxtanuclear localization form of RUSH-IGF2-HA in each experimental group is shown (mean ± SD; n = 3; > 100 cells were counted in each experiment). **, p < 0.01; ***, p < 0.001; ns, not significantly different.

[0024] FIG. 10A to FIG. 10D It is shown that TMED10-FLAG is transported from the ER to the Golgi together with RUSH-IGF2-HA. Fig. 10A and Fig. 10C HeLa cells co-transfected with RUSH-IGF2-HA and TMED10-FLAG, or HeLa cells co-transfected with RUSH-ShhN-HA and TMED10-FLAG are shown. Localization of the indicated proteins was analyzed after 0 min (AC, HJ) or 10 min (DF, KM) biotin treatment on day 1 post-transfection. Scale bar, 10 μm. Fig. 10B and Fig. 10DQuantification of the percentage of cells showing the juxtanuclear localization form of TMED10-FLAG in cells co-expressing TMED10-FLAG and RUSH-IGF2-HA or RUSH-ShhN-HA after indicated times of biotin treatment is shown (mean ± SD; n = 3; > 100 cells were counted in each experiment). ****, p < 0.0001; ns, not significantly different.

[0025] FIG. 11A to FIG. 11C Shown is the analysis of co-localization between RUSH-IGF2-HA and TMED10-FLAG using a permeabilized cell assay. Fig.11A HeLa cells co-transfected with RUSH-IGF2-HA and TMED10-FLAG are shown. On day 1 after transfection, cells were permeabilized with digitonin and then incubated with rat liver cytosol, biotin, and GTPγS at 37°C for 15 min. After incubation, the localization of the indicated proteins was analyzed by immunofluorescence. Scale bar, 10 μm. Fig. 11B and Fig. 11C Shows Fig.11A Magnified view of the indicated area in Image C.

[0026] Fig. 12A and Fig. 12B It was shown that residues 112-140 in IGF2 are sufficient to promote ER to Golgi trafficking. Fig. 12A HeLa cells transfected with plasmids encoding the indicated RUSH constructs were incubated with biotin for the indicated time periods. The localization of the indicated proteins was analyzed by immunofluorescence. Scale bar, 10 μm. Fig. 12B Quantification of the percentage of cells showing juxtanuclear localization of the RUSH constructs after 20 min of biotin treatment is shown (mean ± SD; n = 3; > 100 cells were counted in each experiment).

[0027] A brief description of the sequence

[0028] SEQ ID NO: 1 5'-GTGAGGAGATTCACAAGGA-3' (target sequence for human TMED10).

[0029] SEQ ID NO: 2 5'-GTCCTGTACTTCAGCATCT-3' (target sequence for mouse TMED10).

[0030] SEQ ID NO: 3 5'-GCACAATCTTTACCTCAGA-3' (target sequence for sortilin).

[0031] SEQ ID NO: 4 5'-TAACGGAAAAGGGCCGCGCC-3' (sgRNA targeting exon 1 of TMED10).

[0032] SEQ ID NO: 5 KFFQYDTWKQSTQRLRRGLPALLRARRGHC (synthetic IGF2 112 - 140 peptide).

[0033] SEQ ID NO: 6

[0034] MGIPMGKSMLVLLTFLAFASCCIAAYRPSETLCGGELVDTLQFVCGD

[0035] RGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSERDVS

[0036] TPPTVLPDNFPRYPVGKFFQYDTWKQSTQRLRRGLPALLRARRGHV

[0037] LAKELEAFREAKRHRPLIALPTQDPAHGGAPPEMASNRK

[0038] SEQ ID NO: 7

[0039] MGIPVGKSMLVLLISLAFALCCIAAYGPGETLCGGELVDTLQFVCSD

[0040] RGFYFSRPSSRANRRSRGIVEECCFRSCDLALLETYCATPAKSERDVS

[0041] TSQAVLPDDFPRYPVGKFFQYDTWRQSAGRLRRGLPALLRARRGR

[0042] MLAKELKEFREAKRHRPLIVLPPKDPAHGGASSEMSSNHQ

[0043] SEQ ID NO: 8

[0044] MGITAGKSMLALLAFLAFASCCYAAYRPSETLCGGELVDTLQFVCG

[0045] DRGFYFSRPSSRINRRSRGIVEECCFRSCDLALLETYCAAPAKSERDV

[0046] SASTTVLPDDFTAYPVGKFFQSDTWKQSTQRLRRGLPAFLRARRGR

[0047] TLAKELEALREAKSHRPLIALPTQDPATHGGASSEASSD

[0048] SEQ ID NO:9

[0049] MCAARQILLLLLAFLAYALDSAAAYGTAETLCGGELVDTLQFVCGD

[0050] RGFYFSRPVGRNNRRINRGIVEECCFRSCDLALLETYCAKSVKSERD

[0051] LSATSLAGLPALNKESFQKPSHAKYSKYNVWQKKSSQRLQREVPGI

[0052] LRARRYWQAEGLQAAEEARAMHRPLISLPSQRPPAPRASPEATGPQ

[0053] E

[0054] SEQ ID NO:10

[0055] MEQLSCKHRSSSVEAEAQLCRQTESRSTQLPRMSVMRHLFLLSITFL

[0056] VYTLDSAKAYRATETLCGGELVDTLQFVCGDRGFYFSTNNGRSNRR

[0057] PNRGIVDVCCFKSCDLELLETYCAKPTKNERDVSTAPATAIPPLSKQ

[0058] DLYHKHHHTKSSKYDIWQRKSIHRLRRGVPAIVRARQYRLLMEKAE

[0059] EAEQALSHRPLTTLPITRPLRLQQASEPSHN

[0060] SEQ ID NO:11

[0061] MDDYHVFCASCRKTEETRTTMRSLIVFVLSLSMLISNVTAGETLCGG

[0062] ELVDTLQFVCGEDGFYISRPNRSNSRRPQRGIVEECCFRSCELHLLQQ

[0063] YCAKPVKSERDVSSTSLQVFPVSQALHKDTINVKYSKYEVWQQKA

[0064] AQRLRRGVPSILLARKFRRQMEKIQDEEQTSFHRPLMTLPNRQPAIV

[0065] PHVQISTSRK DETAILED DESCRIPTION

[0066] Selected Definition

[0067] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, when the terms "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising." Transitional Terms / Phrase

[0068] “comprising, comprises, comprise,” “consisting essentially of, consists essentially of,” and “consisting, consists” (and any grammatical variations thereof) may be used interchangeably.

[0069] The phrase “consisting essentially of, consists of

[0070] "Essentially of)" means that the claim includes embodiments containing the specified materials or steps as well as those embodiments that do not materially affect the basic and novel characteristics of the claim.

[0071] The term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured, i.e., the limitations of the measurement system. In the case of compositions containing amounts of ingredients for which the term "about" is used, these compositions have the stated amount of ingredient with a variation (error range) of 0% to 10% (X ±

[0072] 10%). In other cases, the term "about" is used to refer to a variation (error range) of 0% to 10% (X ± 10%) around a given value. Obviously, such a variation represents a range of up to 10% higher or lower than a given value, for example, X ± 1%, X ± 2%, X ±

[0073] 3%, X±4%, X±5%, X±6%, X±7%, X±8%, X±9% or X±10%.

[0074] In the present disclosure, scope is stated in abbreviated form to avoid having to state in detail and describe each value in the scope. Where appropriate, any suitable value in the range can be selected as the upper limit, lower limit or end value of the range. For example, the range of 0.1 to 1.0 represents the end value is 0.1 and 1.0, and the median is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and all intermediate ranges included in 0.1 to 1.0, such as 0.2 to 0.5, 0.2 to 0.8 and 0.7 to 1.0 etc. It is expected that there are at least two significant figures in a range, for example, the range of 5 to 10 represents the whole values ​​between 5.0 and 10.0 and between 5.00 and 10.00, including end value. When using scope in this article, the combination and sub-combination (for example, the sub-range in the disclosed range) of the scope and the specific embodiments thereof are clearly included.

[0075] As used herein, the term "subject" refers to an animal in need or desire of delivering the benefits provided by a therapeutic compound. As used herein, the term "animal" may be, for example, a human, pig, horse, goat, cat, dog, ape, guinea pig, hamster, cattle, or sheep. These benefits may include, but are not limited to, treating a health condition, disease, or disorder; preventing a health condition, disease, or disorder; immune health; enhancing the function of an organ, tissue, or system in the body. The preferred subject in the context of this application is a human. The subject may be at any age or developmental stage, including an infant, toddler, teenager, adult, or elderly.

[0076] As used herein, the term "treat" refers to eradicating, alleviating, ameliorating or reversing the signs or symptoms of a health condition, disease or disorder to any extent, and includes but does not require complete cure of the condition, disease or disorder. Treatment can be curing, ameliorating or partially ameliorating the condition. "Treatment" can also include improving or enhancing a condition or feature, for example, bringing the function of a particular system in the body into an enhanced state of health or homeostasis.

[0077] By "reduced" is meant a negative change of at least 1%, 5%, 10%, 25%, 50%, 75% or 100%.

[0078] By "increase" is meant a positive change of at least 1%, 5%, 10%, 25%, 50%, 75% or 100%.

[0079] As used herein, an "isolated" or "purified" compound is substantially free of other compounds. In certain embodiments, the purified compound is at least 60% by weight (dry weight) of the target compound. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the target compound. For example, a purified compound is a compound that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100% (w / w) by weight of the desired compound. Purity is determined by any suitable standard method, such as by column chromatography, thin layer chromatography or high performance liquid chromatography (HPLC) analysis.

[0080] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," "effective amount," and "effective dose" are used to refer to an amount or dose of a compound or composition thereof that, when administered to a subject, is capable of treating or ameliorating a condition, disease, or disorder in a subject, or is capable of enhancing the health or function of an organ, tissue, or body system. In other words, the amount is "therapeutically effective" when administered to a subject. The actual amount will vary depending on many factors, including, but not limited to, the specific condition, disease, or disorder being treated or ameliorated; the severity of the condition; the specific organ, tissue, or body system whose health or function needs to be enhanced; the patient's weight, height, age, and physical condition; and the route of administration.

[0081] As used herein, the terms "prevent," "reduce," "inhibit," "block," "prevent," "alleviate," or "mitigate" when referring to a compound mean that the compound reduces the incidence, severity, size, volume, or associated symptoms of cancer and / or cancerous lesions by at least about 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 100% compared to how the cancer or cancerous lesions would normally exist without administration of the compound or a composition comprising the compound.

[0082] The recitation of a list of chemical groups in the definition of any variable herein includes defining the variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0083] As used herein, the term "pharmaceutically acceptable" means compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.

[0084] As used herein, the terms "oligonucleotide" and "oligonucleotide" are used interchangeably to describe a short single strand of synthetic DNA or RNA, for example, a sequence of about 5 to about 500 nucleic acid bases.

[0085] As used herein, "vector" refers to a DNA molecule, such as a plasmid, used to introduce a nucleotide construct (e.g., a DNA construct) into a host cell. Cloning vectors typically contain one or a small amount of restriction endonuclease recognition sites and marker genes suitable for identifying and selecting cells transformed with the cloning vector, wherein the restriction endonuclease recognition sites can be inserted into exogenous DNA sequences in a determinable manner without losing the basic biological function of the vector. Marker genes typically include genes that provide selective characteristics such as tetracycline resistance, hygromycin resistance, or ampicillin resistance.

[0086] In this application, the terms "peptide", "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0087] The term "label" and similar terms refer to a component detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful labels include fluorescent dyes (fluorophores), luminescent agents, electron-dense reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin, enzymes acting on substrates (e.g., horseradish peroxidase), digoxigenin, 32 P and other isotopes, haptens and proteins. The term includes combinations of single labeling agents, for example, combinations of fluorophores that provide unique detectable labels at a specific wavelength or combination of wavelengths. In the case of detecting nucleic acids (e.g., target sequences), probes can typically be labeled with radioactive isotopes, fluorescent markers (fluorophores), or luminescent agents.

[0088] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0089] Other features and advantages of the present application will be apparent from the following description of preferred embodiments of the application and from the claims.

[0090] All references cited herein are incorporated by reference in their entirety.

[0091] The present application relates to novel compositions and methods for blocking the interaction between TMED10 and IGF2, wherein the blocking is achieved by mutating residues 112-140 on IGF2 or mutating the GOLD motif on TMED10, or utilizing a polypeptide comprising the GOLD domain of human TMED10, or a chemical small molecule that blocks the interaction between TMED10 and IGF2 as an effective way to inhibit IGF2 secretion, thereby inhibiting downstream signaling. The p24 family cargo receptor TMED10 regulates IGF2 secretion by recognizing the export motif on IGF2. Detailed mechanistic analysis reveals a direct interaction between the GOLD domain of TMED10 and residues 112-140 of IGF2.

[0092] In certain embodiments, blocking the interaction between TMED10 and IGF2 provides a new therapeutic strategy to inhibit IGF2 signaling, thereby stopping the progression of cancer or neurodevelopmental disorders associated with IGF2 dysregulation. In fact, the binding motifs of TMED10 and IGF2 are critical for the interaction between IGF2 and TMED10. In certain embodiments, mutations in these key motifs can be used as a new method to manipulate IGF2 secretion for cancer treatment. In addition, sortilin, a single-pass transmembrane protein from the vacuolar protein sorting 10 protein family, mediates post-Golgi transport of IGF2.

[0093] Existing IGF2 antagonists focus on blocking the interaction between IGF2 and its receptors such as IGFLR. Although IGF2 antagonists show potential therapeutic significance, it is challenging to achieve sufficient and sustained efficacy in inhibiting IGF2 signal transduction. Due to the similarity between IGF2 and IGF1 or IL family proteins, specificity is also a problem. Specifically, effective antagonists should selectively bind to IGF2 without interfering with the function of other related growth factors or receptors (such as insulin receptors). However, there is no antagonist specific for the intracellular transport of IGF2.

[0094] Dysregulation of the IGF2 signaling pathway is associated with a variety of conditions. IGF2 is known to play a role in muscle growth and development and has been studied in the context of muscle regeneration and repair. Blocking the secretion of IGF2 can lead to defects in muscle stem cell differentiation. TMED10 mediates the secretion of IGF2 and further regulates C2C12 differentiation in an autocrine manner. In certain embodiments, blocking IGF2 secretion by inhibiting the interaction between TMED10 and IGF2 is an effective way to downregulate IGF2 signaling and block cancer progression.

[0095] The secretion of IGF2 depends on a direct interaction between residues 112-140 of IGF2 and TMED10. TMED10 acts as a cargo receptor to mediate the ER export of IGF2 for myoblast differentiation. The GOLD domain of TMED10 is also critical for IGF2 secretion. In addition, TMED10 regulates the ER export of sortilin, and sortilin is important for the TGN export of IGF2.

[0096] After IGF2 is synthesized by the ribosome, it needs to be delivered along the secretory transport pathway to exert its physiological function. IGF2 is first synthesized as a precursor hormone containing 180 amino acids. After entering the ER, the N-terminal signal peptide is cleaved to produce pro-IGF2 (IGF2 25-180 ). The correctly folded pro-IGF2 protein is then packaged into transport vesicles for delivery to the Golgi apparatus. Pro-IGF2 undergoes O-glycosylation and endoproteolysis in the Golgi apparatus to produce the IGF2 peptide IGF2 25-128 , IGF2 25-111 and mature IGF2 (IGF2 2-91 ).

[0097] Capsid protein complex II (COPII) is a key player in regulating the packaging of cargo proteins into vesicles in the ER. In the conventional secretory transport pathway, soluble cargo proteins in the ER lumen cannot be directly recognized by the COPII capsid; instead, it is believed that these cytoplasmic proteins are transported under the recognition of transmembrane cargo receptors. ERGIC53 is a major cargo receptor that recruits a variety of soluble cargo proteins to COPII vesicles in mammals. In addition, the p24 protein plays a key role in ER-Golgi bidirectional transport.

[0098] In certain embodiments, provided herein is a pharmaceutical composition for inhibiting IGF2 secretion by inhibiting the interaction between IGF2 and TMED10 in cells of a subject exhibiting aberrant IGF2 signaling.

[0099] In other embodiments, provided herein is a method for treating a subject with abnormal IGF2 signaling, the method being achieved by providing a pharmaceutical composition comprising an inhibitor of IGF2-TMED10 interaction in a cell and one or more pharmaceutical carriers or excipients, and administering an effective amount of the pharmaceutical composition to the subject. In preferred embodiments, the inhibitor comprises: a human IGF2 polypeptide comprising residues 112-140 of the human IGF2 sequence; or a human TMED10 polypeptide comprising residues 1-130 of the human TMED10 sequence; and combinations thereof. In certain embodiments, the inhibitor comprises: a human TMED10 polypeptide comprising residues 32-132 of the GOLD domain of the human TMED10 sequence. In certain embodiments, the inhibitor includes, but is not limited to, a small molecule, a covalent inhibitor or an antibody and combinations thereof.

[0100] In other embodiments, provided herein is a method for treating cancer in a subject in need thereof, the method being achieved by providing a pharmaceutical composition comprising an inhibitor of IGF2-TMED10 interaction in a cell and one or more pharmaceutical carriers or excipients, and administering an effective amount of the pharmaceutical composition to the subject. In preferred embodiments, the inhibitor comprises: a human IGF2 polypeptide comprising residues 112-140 of the IGF2 sequence; or a human TMED10 polypeptide. It comprises residues 1-130 of the human TMED10 sequence; and combinations thereof. In certain embodiments, the inhibitor comprises residues 120-140 of the IGF2-TMED10 sequence. In other embodiments, the inhibitor comprises residues 125-140 of the IGF2-TMED10 sequence. In certain embodiments, the inhibitor includes, but is not limited to, small molecules, covalent inhibitors or antibodies and combinations thereof.

[0101] In preferred embodiments, the compositions and methods of the present application inhibit the secretion of IGF2 in cells having aberrant IGF2 signaling.

[0102] In certain embodiments, the nucleotide sequences of SEQ ID No: 1, 2, 3 and 4 also include sequences that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75% and 70% homologous. In certain embodiments, the amino acid sequence of peptide SEQ ID No: 5 also includes sequences that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75% and 70% homologous.

[0103] Carriers and / or excipients according to the present application may include any and all solvents, diluents, buffers (e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffer), oil-in-water or water-in-oil emulsions, aqueous compositions suitable for (e.g.) IV use with or without organic co-solvents, solubilizers (e.g., polysorbate 65, polysorbate 80), colloids, dispersion media, vehicles, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, thickeners (e.g., carbomer, gelatin or sodium alginate), coatings, preservatives (e.g., thimerosal, benzyl alcohol, polyquaternium salts), antioxidants (e.g., ascorbic acid, sodium metabisulfite), tonicity control agents, absorption delaying agents, adjuvants, bulking agents (e.g., lactose, mannitol), and the like. In addition, fillers, anticoagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives, etc. may also be included. The use of carriers and / or excipients in the field of medicine and supplements is well known. In addition to any conventional media or agents that are incompatible with the target health-promoting substance or with the adjuvant composition, it is contemplated that a carrier or excipient may be used in the composition of the present application.

[0104] In one embodiment, the pharmaceutical composition can be formulated for use by injection, for example, as solution or suspension. The route of administration of the pharmaceutical composition of the present application may also include but is not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, enteral, local, sublingual or rectal routes. The term "parenteral" refers to include subcutaneous, percutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intradural, intralesional and intracranial injection or infusion technology. The pharmaceutical composition of the present application may also be formulated into suppositories for use in the rectum.

[0105] The solution or suspension may contain: a suitable non-toxic, parenterally acceptable diluent or solvent, such as mannitol, 1,3-butylene glycol, water, Ringer's solution or isotonic sodium chloride solution; or a suitable dispersant or wetting agent and suspending agent, such as sterile, non-irritating, fixed oils, including synthetic mono- or di-glycerides and fatty acids, including oleic acid. An illustrative example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and the balance USP water for injection (WFI). Other illustrative carriers for intravenous use include: 10% USP ethanol and USP WFI; 0.01% to 0.1% triethanolamine USP WFI solution; 0.01% to 0.2% dipalmitoyl diphosphatidylcholine USP WFI solution; and 1% to 10% squalene or parenteral vegetable oil in water-in-oil emulsions. Water or saline solutions and aqueous glucose and glycerol solutions can be preferably used as carriers, particularly for injectable solutions. Illustrative examples of carriers for subcutaneous or intramuscular use include phosphate buffered saline (PBS) solutions, 5% glucose in WFI solutions and 0.01% to 0.1% triethanolamine in 5% glucose solutions or 0.9% sodium chloride in USP WFI solutions; or a 1:2 or 1:4 mixture of 10% USP ethanol, 40% propylene glycol and the balance being an acceptable isotonic solution such as 5% glucose or 0.9% sodium chloride; or 0.01% to 0.2% dipalmitoyl diphosphatidylcholine in USP WFI solutions and 1% to 10% squalene or parenteral vegetable oils in water-in-oil emulsions.

[0106] The pharmaceutical composition of the present application may vary according to a variety of factors, including the activity of a certain active ingredient used, the age, weight, general health, sex, diet, administration time, route of administration, excretion rate, drug combination and the severity of a certain disease to be prevented or treated. The dosage of the pharmaceutical composition may vary according to the patient's condition, weight, severity of the disease, drug form, route of administration and duration, and may be appropriately selected by those skilled in the art. Preferably, taking into account all of the above factors, an amount that can achieve the maximum effect with a minimum amount without side effects can be applied, and more preferably, an effective dose of 0.1 mg / body weight kg / day to 1,000 mg / body weight kg / day, even more preferably 0.1 mg / body weight kg / day to 10 mg / body weight kg / day can be repeated several times a day. The above dosage does not limit the scope of the present application in any way.

[0107] In some embodiments of the present application, the method includes applying multiple doses of the compound of the present application. The method may include applying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more therapeutically effective doses of the composition comprising the compound of the present application as described herein. In some embodiments, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days, 2 months, 3 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, 2.5 years, 5 years or more than 10 years of application dosage. The frequency of application and duration of the multi-dose composition are (for example) to prevent or treat nephropathy or inflammatory reaction. In addition, treating a subject with a therapeutically effective amount of the compound of the present application may include a single treatment or may include a series of treatments. It should also be understood that the effective dose of the compound for treatment may increase or decrease during a specific treatment. Changes in dosage may occur and become more apparent in test results of kidney disease (e.g., glomerular filtration rate).In some embodiments of the present application, the method comprises administering the compound several times a day, including but not limited to 2 times a day, 3 times a day, and 4 times a day.

[0108] In some embodiments of the present application, the method includes administering multiple doses of the composition of the present application. The method may include administering a composition of a compound of the present application as described herein or a composition thereof once a day, once a week, once a month, once a quarter, twice a year, once a year, or less frequently. In addition, treating a subject with a therapeutically effective amount of the composition of the present application may include a single treatment or may include a series of treatments. It should also be understood that the effective dose of the compound or its composition for treatment may increase or decrease during a specific treatment. Changes in dosage may occur and become more apparent in the diagnostic analysis results known in the art to determine the presence of cancer cells.

[0109] In one embodiment, the cell is a cancer cell or tumor cell selected from, for example, a prostate cancer cell, a gallbladder cancer cell, an intrahepatic bile duct cancer cell, a bile duct cancer cell, an oral cancer cell, a pharyngeal cancer cell, a laryngeal cancer cell, a tongue cancer cell, a duodenal cancer cell, an eye tumor cell, a mediastinal cancer cell, a sinus cancer cell, a renal pelvis cancer cell, a cardiac cancer cell, a glioblastoma cell, a neuroblastoma cell, a liver cancer cell, a bone cancer cell, a pancreatic cancer cell, a skin cancer cell, a head and neck cancer cell, a breast cancer cell, a lung cancer cell, a skin or intraocular malignant melanoma cell, a renal cancer cell, a uterine cancer cell, an ovarian cancer cell, a colon cancer cell, a rectal cancer cell, an anal region cancer cell, a colorectal cancer cell, a gastric cancer cell, a testicular cancer cell, a fallopian tube cancer endometrial cancer cell, a cervical cancer cell, a vaginal cancer cell. cells, vulvar cancer cells, esophageal cancer cells, small intestinal cancer cells, endocrine system cancer cells, thyroid cancer cells, parathyroid cancer cells, adrenal cancer cells, soft tissue sarcoma cells, urethral cancer cells, penile cancer cells, childhood cancer cells, lymphoma cells, bladder cancer cells, ureteral cancer cells, renal pelvis cancer cells, central nervous system (CNS) cancer cells, primary CNS lymphoma cells, spinal cord cancer cells, brain stem glioma cells, pituitary adenoma cells, Kaposi's sarcoma cells, epidermal cancer cells, squamous cell carcinoma cells, follicular lymphoma cells, immune large cell lymphoma cells, mantle cell lymphoma cells, mycosis fungoides cells, hepatoblastoma cells, retinoblastoma cells, peritoneal cancer cells, brain tumor cells, thymic cancer cells, and any combination of the above cancer cells.

[0110] In some embodiments of the present application, the method includes activating the compound of the present application by irradiation with light. The method may include irradiating the cancer cells treated with the composition of the present application 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or more. In some embodiments, the cancer is irradiated over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5 years, about 5 years, or more than 10 years. In addition, irradiation treatment of a subject for cancer may include a single treatment or may include a series of treatments. It should also be understood that the effective excitation frequency, intensity, and duration for treatment may increase or decrease during a particular treatment. Changes in excitation frequency, intensity, and duration may occur and become more apparent in test results of cancer remission known in the art. In some embodiments of the present application, the method comprises irradiating the compound several times a day, including but not limited to 2 times a day, 3 times a day, and 4 times a day. In a preferred embodiment, irradiating at least one dose of the composition is repeated every day for about 2 weeks to about 10 weeks.

[0111] In some embodiments, the cancer or tumor targeted by the present application is in any stage in a variety of stages, including newly diagnosed, recurrent, refractory, progressive disease, relief and other. In some embodiments, the cancer or tumor treated is a newly diagnosed cancer. In some embodiments, the cancer or tumor is a recurrent cancer (for example, recurrent gynecological cancer, such as recurrent epithelial ovarian cancer, recurrent fallopian tube cancer, recurrent primary peritoneal cancer or recurrent endometrial cancer). In some embodiments, the present application is applicable to the treatment of metastatic cancer.

[0112] In certain embodiments, the composition of the present application of a therapeutically effective amount can be administered intraperitoneally or by a sustained release system, and a sustained release system is for example a semipermeable matrix of a solid hydrophobic polymer containing a compound of the present application. Administration can also be by other carriers or vehicles, such as patches, micelles, liposomes, vesicles, implants (e.g., microimplants), synthetic polymers, microspheres, nanoparticles, etc. In certain embodiments, nanoparticles can be used to administer the composition so that the composition passes through the skin.

[0113] The composition may further comprise a preparaton, such as a suspending agent, a stabilizing agent and / or a dispersing agent. In a further embodiment, the active ingredient of the composition according to the present application may be in powder form, obtained by aseptic isolation of sterile solids or by lyophilization from a solution, for preparation with a suitable vehicle (e.g., sterile, pyrogen-free water) prior to use.

[0114] In one embodiment, the composition of the present application can be formulated for application on the skin by topical application, for example, as a topical composition, it includes flushing agents, sprays or drops, lotions, gels, ointments, creams, foams, powders, solids, wipes, tapes, steam agents, pastes, tinctures, or transdermal patches. In addition to any pharmaceutically active carrier, suitable formulations for topical application can include, for example, softeners, such as carnauba wax, cetyl alcohol, cetyl ester wax, emulsifying wax, aqueous lanolin, lanolin alcohol, microcrystalline wax, paraffin, vaseline, polyethylene glycol, stearic acid, stearyl alcohol, white beeswax or yellow beeswax. In addition, the composition can include: a wetting agent, such as glycerol, propylene glycol, polyethylene glycol, sorbitol solution and 1,2,6-hexanetriol; or a penetration enhancer, such as ethanol, isopropanol or oleic acid.

[0115] Regardless of the selected route of administration, the composition can be formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. Similar to other drugs, the composition can be formulated for administration in any convenient manner, for use in human or veterinary medicine.

[0116] The composition may further comprise one or more pharmaceutically acceptable carriers and / or excipients, and may be formulated into a preparation, for example, a semisolid or liquid form, such as a solution or an injection.

[0117] The preparation can be conveniently presented in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of the compound that can be combined with a carrier material to produce a single dosage form will vary according to the subject being treated, the specific mode of administration. The amount of the active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of the compound that produces the therapeutic effect. Typically, in one hundred percent, the amount ranges from about 1% by weight to about 99% by weight of the active ingredient, preferably from about 5% by weight to about 70% by weight, and most preferably from about 10% by weight to about 30% by weight.

[0118] As long as there is no contradiction, the above contents of the present application are equally applicable to each other, and those skilled in the art can implement them through appropriate changes, which are also included in the scope of the present application.

[0119] Hereinafter, the present application will be described in detail through examples, but the scope of the present application is not limited to the following examples.

[0120] Materials and methods

[0121] Constructs, reagents, cell culture, immunofluorescence, and transfection

[0122] The cDNA encoding human IGF2, human TMED10, human sortilin and the plasmid encoding Str-KDEL_SBP-EGFP-HA-IGF2 were synthesized by BGI (Beijing, China). The plasmids encoding IGF2 with 3xHA tag (IGF2-HA), IGF2 with GST tag, TMED10 with 3xFLAG tag (TMED10-FLAG), Str-KDEL_SBP-EGFP-sortilin, Str-KDEL_SBP-EGFP-sortilin-myc, Str-KDEL_SBP-EGFP-IGF2-HA and truncated forms of IGF2 were generated by standard molecular cloning methods. Plasmids encoding mutant forms of IGF2 were generated by QuikChange II site-directed mutagenesis using plasmids encoding IGF2-HA or Str-KDEL_SBP-EGFP-IGF2-HA as templates. A plasmid encoding siRNA-resistant TMED10-FLAG was generated by QuikChange II site-directed mutagenesis using the plasmid encoding TMED10-FLAG as a template.

[0123] siRNA against TMED10 was purchased from Ribo-bio (Guangzhou, China). The target sequence for human TMED10 is GTGAGGAGATTCACAAGGA. The target sequence for mouse TMED10 is GTCCTGTACTTCAGCATCT. The target sequence for sortilin is GCACAATCTTTACCTCAGA. Commercially available antibodies were rabbit anti-HA (Cell Signaling, catalog number 3724), rabbit anti-TMED10 (Proteintech, catalog number 15199-1-AP), mouse anti-MHC (DSHB, catalog number MF 20), and mouse anti-myogenin (Santa Cruz Biotechnology, catalog number SC-12732); sheep anti-TGN46 (BIO-RAD, catalog number AHP500G), mouse anti-GM130 (BD Bioscience, catalog number 610823); mouse anti-FLAG (Sigma, catalog number F3165); mouse anti-β-actin antibody (Proteintech, catalog number 60008-1-Ig); goat anti-GST (GE Healthcare, catalog number 27-4577-01); mouse anti-TMED2 (Santa Cruz Biotechnology, catalog number SC-376459); mouse anti-Myc (Cell Signaling, catalog number 27-4577-01); goat anti-GST (GE Healthcare, catalog number 27-4577-01); mouse anti-TMED2 (Santa Cruz Biotechnology, catalog number SC-376459); mouse anti-Myc (Cell Signaling, catalog number 27-4577-01); mouse anti- Signaling, catalog number 2276); rabbit anti-sortilin (Proteintech, catalog number 12369-1-AP). Rabbit anti-Sec22B antibody and rabbit anti-ERGIC53 antibody were kindly provided by Professor Randy Schekman (University of California, Berkeley, CA, USA). Rabbit anti-TMED7 antibody was kindly provided by Professor Pingbo Huang (Hong Kong University of Science and Technology, Hong Kong SAR, China).

[0124] HeLa cells and HEK293T cell lines are provided by the cell culture facility of the University of California, Berkeley, and are confirmed by short tandem repeat analysis. Mycoplasma contamination detection of all cell lines is negative. HeLa and HEK293T cells are cultured in the Eagle medium (DMEM) modified by Dulbecco containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture (Invitrogen). C2C12 cells are cultured in the Eagle medium (DMEM) modified by Dulbecco containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin mixture (Invitrogen). As described in AJPing et al. (referring to document AJPing et al., Genetic linkage of Beckwith-Wiedemann syndrome to 11p15.Am J Hum Genet 44,720-723 (1989)), siRNA or DNA constructs are transfected into HeLa cells or HEK293T cells and immunofluorescence is performed. Images were acquired using a Zeiss AxioObserver Z1 microscope system (Carl Zeiss, Germany) equipped with an ORCA Flash 4.0 camera (Hamamatsu, Japan) or a Leica SP8 confocal microscope (Leica, Germany).

[0125] For CRISPR experiments, sgRNA sequences were ligated into the AAV9-U6-Dsred plasmid. Clonal cell lines were obtained by diluting the cell suspension to a single cell per well and amplifying each well. Genotyping of clonal cell lines was performed by Sanger sequencing of target site PCR amplicons of genomic DNA isolated by the Puregene kit (Qiagen). The sgRNA was as follows: TMED10, 5'TAACGGAAAAGGGCCGCGCC' targeting exon 1 of TMED10.

[0126] Retention on Selective Hook (RUSH) Test

[0127] The RUSH assay was performed as described by NJ Samani et al. (see NJ Samani et al., Genomewide association analysis of coronary artery disease. N Engl J Med 357, 443-453 (2007)). In brief, HeLa cells were transfected with plasmids encoding different forms of RUSH constructs of Str-KDEL and SBP-EGFP-sortilin or SBP-EGFP-sortilin-myc or IGF2. On the first day after transfection, the cells were incubated for 2 hours in complete medium containing 100 ng / μl cycloheximide. Then, the cells were incubated for the specified time in complete medium containing 40 μM biotin (Sigma-Aldrich) and 100 ng / μl cycloheximide (Sigma-Aldrich). Subsequently, the cells were fixed with 4% PFA and fixed on a slide with DAPI (Invitrogen) by ProLongTM Gold Antifade Mountant for microscopic analysis.

[0128] To analyze the secretion of IGF2, HeLa cells transfected with plasmids encoding RUSH constructs of different forms of Str-KDEL and IGF2 were incubated in complete medium containing 100 ng / μl cycloheximide for 2 hours. The cells were then treated with 100 ng / μl cycloheximide and 40 μM biotin-free FBS medium for the indicated time. The secreted proteins were then precipitated by TCA precipitation. The cells were collected and lysed with HKT buffer (100 mM KCl, 20 mM Hepes, pH 7.2, 0.5% Triton X-100). Secreted proteins and cell lysates were analyzed by immunoblotting.

[0129] Immunoprecipitation, protein purification, and binding assays

[0130] FLAG-tagged TMED10 or TMED10 were expressed by treating HEK293T cells co-transfected with plasmids encoding the indicated proteins in PBS containing 2 mM dithiobis[succinimidyl propionate] (DSP) and 2 mM CaCl2 for 30 min at room temperature, followed by quenching with 25 mM Tris-HCl, pH 7.5. 1-130Immunoprecipitation. The cells were then lysed in lysis buffer (50mMTris-HCl, 150mM NaCl, 2mM CaCl2 and 0.1% TX-100, supplemented with protease inhibitors (Roche), pH7.5). Subsequently, 500μl of 0.5mg / ml cell lysate was incubated overnight at 4°C with 10μl of dense anti-FLAG agarose affinity beads. M2-FLAG affinity beads were incubated in blocking buffer (50mM Tris-HCl, 500mM NaCl, 2mM CaCl2, 5% BSA, pH7.5) for 1 hour for pre-blocking to reduce nonspecific binding. After incubation, the beads were washed 3 times with 1ml blocking buffer (5% BSA), and washed 2 times with 1ml blocking buffer (-BSA), and the bound substances were analyzed by immunoblotting.

[0131] GST-tagged TMED10 was performed as described by Y. Guo et al. 1-130 Purification of Vangl2 (see Y. Guo, G. Zanetti, R. Schekman, A novel GTP-binding protein-adaptor protein complex responsible for export of Vangl2 from the trans Golgi network. Elife 2, e00160 (2013)). 1-130 The dense GSH beads were used for GST pull-down assay. The beads were incubated with 200 μl of 0.5 mg / ml of cell lysate in HKT buffer at 4°C overnight with mixing, the cell lysate being derived from HEK293T cells transfected with the indicated plasmids. After incubation, the beads were washed three times with 500 μl of HKT buffer and twice with 500 μl of HK buffer, and the bound material was analyzed by immunoblotting.

[0132] Peptide binding assays were performed as described previously. Synthetic IGF2 112-140 peptide (KFFQYDTWKQSTQRLRRGLPALLRARRGHC) was purchased from GenScript and coupled to thiopyridone-Sepharose 6B beads (Sigma-Aldrich) via an additional C-terminal cysteine ​​residue. For binding experiments, GST or GST-tagged TMED10 was added to 450 μl of binding buffer (20 mM Hepes, pH 7.2, 250 mM sorbitol, 70 mM KOAc, 1 mM Mg(OAc)2, and 1 mg / ml bovine serum albumin). 1-130Pre-incubated at 4°C for 30 minutes with approximately the same abundance (~5 pmol). The coupled beads were blocked by incubation in 5 mM β-mercaptoethanol, 50 mM NaOAc, 0.5 M NaCl at pH 4.5 for 40 minutes at 4°C, followed by a wash step and incubation in binding buffer at 4°C for 2 hours. After incubation, 250 μl of buffer containing approximately 5 μl of beads conjugated with peptides was added to the reaction mixture and kept at 4°C for 80 minutes. The beads were washed five times by incubation with binding buffer containing 0.5 M KOAc and 0.1% Triton but without BSA for 1.5 minutes, followed by washing 3 times with binding buffer containing 0.1% Triton but without BSA. The beads were then analyzed by immunoblotting.

[0133] In vitro vesicle formation assay

[0134] As described by X. Tang et al., an in vitro vesicle release assay was performed (see X. Tang et al., Molecular mechanisms that regulate export of the planar cell-polarity protein Frizzled-6 out of the endoplasmic reticulum. J Biol Chem 10.1074 / jbc. RA120.012835 (2020); X. Tang, F. Yang, Y. Guo, Cell-free Reconstitution of the Packaging of Cargo Proteins into Vesicles at the trans Golgi Network. Bio Protoc 10, e3537 (2020)). In brief, HeLa cells were not transfected or transfected with control siRNA or siRNA for TMED10. On the first day after transfection, cells were transfected with a plasmid encoding RUSH-IGF2-HA. On the second day after knockdown, HEK293T cells were permeabilized on ice for 5 minutes in 3 ml ice-cold KOAc buffer (110 mM potassium acetate, 20 mM Hepes, pH 7.2, 2 mM magnesium acetate) containing 40 mg / ml digitonin. Then, semi-intact cells were precipitated by centrifugation at 300 g for 3 minutes at 4 ° C. The cell pellet was washed twice with 1 ml KOAc buffer and resuspended in 100 μl KOAc buffer. The budding assay was performed by incubating semi-intact cells with 2 mg / ml of rat liver cytosol (about 0.02 OD / reaction) in a 100 μl reaction mixture containing 200 mM GTP and ATP regeneration system (40 mM creatine phosphate, 0.2 mg / ml creatine phosphate kinase and 1 mM ATP) in the presence or absence of 0.5 mg SAR1A (H79G). After incubation at 32°C for 1 hour, the reaction mixture was centrifuged at 14,000 g to remove cell debris and large membranes. The medium speed supernatant was then centrifuged at 100,000 g to precipitate small vesicles. The pellet fraction was then resuspended in 100 μl of 35% OptiPrep and superimposed with 700 μl 30% OptiPrep and 30 μl KOAc buffer. The sample was centrifuged at 55,000 rpm for 2 hours at 4°C in a TLS55 rotor of a Beckman ultracentrifuge. After centrifugation, the top fraction was analyzed by SDS-PAGE and immunoblotting.

[0135] Vesicle formation assays were then performed on a large scale using a 15-cm dish of WT HeLa cells or TMED10 KO HeLa cells to provide donor membranes. Vesicle components were then analyzed by label-free quantitative mass spectrometry using procedures described previously (25).

[0136] Vesicle immunoprecipitation (Vesicle-IP) assay

[0137] In each experimental group of the vesicle-IP assay, one 10 cm dish of transfected or untransfected HeLa cells was used to provide semi-intact cells for the vesicle formation assay. The semi-intact cells were incubated with 2 mg / ml of rat liver cytosol at 32°C for 1 hour in a 1.8 ml reaction mixture containing 200 mM GTP and ATP regeneration system. The reaction mixture was centrifuged at 16,900 g for 20 minutes. The medium-speed supernatant was then rotated overnight at 4°C with 30 μl Pierce anti-HA magnetic beads. The next day, the magnetic beads were collected using a magnetic stand. The supernatant was collected and centrifuged at 100,000 g for 30 minutes at 4°C in a S120AT3 (Hitachi) rotor to precipitate vesicles as "flow-through". The beads were washed 3 times each with ice-cold KOAc buffer containing 1 M, 0.5 M and 0.11 M potassium acetate, respectively. Proteins in the flow-through fraction and the fraction on the beads were analyzed by immunoblotting.

[0138] Muscle stem cell differentiation assay

[0139] Undifferentiated C2C12 cells were cultured with DMEM containing 20% ​​FBS and 1% penicillin-streptomycin mixture. To induce differentiation, C2C12 cells transfected with control siRNA or siRNA against TMED10 were incubated for 3 days in DMEM containing 2% horse serum and 1% penicillin-streptomycin in the presence or absence of 100 ng / ml purified IGF2 (R&D Systems, catalog number: 792-MG). Cells were then analyzed by immunoblotting or immunofluorescence.

[0140] Preparation of samples for mass spectrometry analysis of proteins secreted by C2C12 cells.

[0141] Undifferentiated C2C12 cells grown in 15 cm culture dishes were cultured with DMEM containing 20% ​​FBS and 1% penicillin-streptomycin mixture until the cells reached 80% to 90% confluence. Then, C2C12 cells were transfected with siRNA for GFP or siRNA for mouse TMED10. On the first day after transfection, cells were washed five times with PBS and then cultured for 3 days in DMEM containing 1% penicillin-streptomycin mixture. Culture medium was collected and cell debris was removed by centrifugation. Subsequently, TCA precipitation was performed to precipitate secreted proteins from the culture medium. Precipitated proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue (Bio-SafeTM Coomassie-G250) staining. Subsequently, in-gel digestion and label-free mass spectrometry were performed as described by NJ Samani et al. (see above-mentioned document NJ Samani et al.).

[0142] All patents, patent applications, provisional applications, and publications referenced or cited herein are hereby incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0143] The following are examples of the process of implementing the present invention. These examples should not be construed as limiting. Unless otherwise stated, all percentages are weight percentages, and all solvent mixing ratios are volume ratios.

[0144] Example 1—TMED10 mediates ER-to-Golgi trafficking and secretion of IGF2

[0145] Insulin-like growth factor 2 (IGF2) plays a key role in cell proliferation, migration, differentiation and survival. Despite its importance, the molecular mechanisms that mediate the trafficking of IGF2 along the secretory pathway remain unclear. Newly synthesized IGF2 needs to be delivered along the secretory transport pathway to exert its physiological functions. IGF2 is first synthesized as a precursor hormone containing 180 amino acids. After transport into the ER, the N-terminal signal peptide is cleaved to produce pro-IGF2 (IGF2 25-180 ). The correctly folded pro-IGF2 protein is then packaged into transport vesicles for delivery to the Golgi apparatus. At the Golgi apparatus, pro-IGF2 undergoes O-glycosylation modification and endoproteolytic cleavage to produce the IGF2 peptide IGF2 25-128 , IGF2 25-111 and mature IGF2 (IGF2 25-91) (See SJ Duguay et al., Post-translational processing of the insulin-like growth factor-2 precursor. Analysis of O-glycosylation and endoproteolysis. J Biol Chem 273, 18443-18451 (1998)). Capsid protein complex II (COPII) is a key player in regulating the packaging of cargo proteins into vesicles at the ER. In conventional secretory transport pathways, soluble cargo proteins in the ER lumen cannot be directly recognized by the COPII capsid; instead, it is believed that these cytoplasmic proteins are transported under the recognition of transmembrane cargo receptors (see J. Dancourt, C. Barlowe, Protein sorting receptors in the early secretory pathway. Annu Rev Biochem 79, 777-802 (2010)). ERIGIC53 is a major cargo receptor that recruits a variety of soluble cargo proteins to COPII vesicles in mammals (see the above-mentioned J. Dancourt et al.). In addition, p24 protein plays a key role in ER-Golgi bidirectional transport.Some p24 family members act as cargo receptors to regulate the ER export of specific GPI-anchored proteins and autotaxins in mammalian cells (see J. Dancourt, C. Barlowe, Protein sorting receptors in the early secretory pathway. Annu Rev Biochem 79, 777-802 (2010); L. Lyu et al., Selective export of autotaxin from the endoplasmic reticulum. J Biol Chem 292, 7011-7022 (2017)) and mediate the secretion of Wnt proteins in Drosophila (see T. Buechling, V. Chaudhary, K. Spirohn, M. Weiss, M. Boutros, p24 proteins are required for secretion of Wnt ligands. EMBO Rep 12, 1265-1272 (2011; X. Li et al., Drosophila p24 and Sec22 regulate Wingless trafficking in the early secretory pathway. Biochem Biophys Res Commun 463, 483-489 (2015)). It has also been proposed in the art that soluble cargo proteins enter the newly formed COPII vesicles by default, and this process is called bulk flow (see C. Barlowe, A. Helenius, Cargo Capture and Bulk Flow in the Early Secretory Pathway. Annu Rev Cell Dev Biol 32, 197-222 (2016)).

[0146] The trans-Golgi network (TGN) is another important site in the secretory transport pathway. At the TGN, various cargo adaptors and receptors have been shown to capture cargo molecules into nascent vesicles (see Y. Guo, DW Sirkis, R. Schekman, Protein sorting at the trans-Golgi network. Annu Rev Cell Dev Biol 30, 169-206 (2014)). Sortilin is one of the cargo receptors localized in the Golgi apparatus. It is essential for the sorting of many proteins in the anterograde and retrograde pathways and is widely involved in a variety of physiological activities, including lipid metabolism, neuronal development, immune system, myogenesis and diabetes (see S. Ouyang, B. Jia, W. Xie, J. Yang, Y. Lv, Mechanism underlying the regulation of sortilin expression and its trafficking function. J Cell Physiol 235, 8958-8971 (2020); DM Conlon, Role of sortilin in lipid metabolism. Curr Opin Lipidol 30, 198-204 (2019); SY Xu, J. Jiang, A. Pan, C. Yan, XX Yan, Sortilin: a new player in dementia and Alzheimer-type neuropathology. Biochem Cell Biol 96, 491-497 (2018); H. Talbot et al. al.,RegulatoryRoles of Sortilin and SorLA in Immune-Related Processes.Front Pharmacol 9,1507(2018);M.Ariga,T.Nedachi,H.Katagiri,M.Kanzaki,Functional role of sortilin myogenesis and development of insulin-responsive glucose transportsystemin C2C12 myocytes.J Biol Chem 283,10208-10220(2008)).In particular, non-coding genetic variants at a locus near the gene encoding sortilin are significantly associated with human LDL cholesterol and coronary artery disease (see TM Teslovich et al., Biological, clinical and population relevance of 95 loci for blood lipids. Nature 466, 707-713 (2010); the above-mentioned NJ Samani et al.). Further analysis showed that sortilin interacts with apolipoprotein B100 (ApoB100) and regulates the secretion of lipoproteins containing ApoB100 in hepatocytes, but whether sortilin upregulates or downregulates lipoprotein secretion remains controversial (see K. Musunuru et al., From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus. Nature 466, 714-719 (2010); M. Kjolby et al., Sort1, encoded by the cardiovascular risk locus 1p13.3, is a regulator of hepatic lipoprotein export. Cell Metab 12, 213-223 (2010)).

[0147] The secretion of newly synthesized soluble signaling proteins from producer cells is tightly coupled to downstream signaling pathways in target cells. Although a series of factors regulating the expression of IGF2 have been identified, such as mTOR, PLD1 and miR-125b (see the above-mentioned literature Y. Ge et al.; literature E. Erbay, IH Park, PD Nuzzi, CJ Schoenherr, J. Chen, IGF-II transcription in skeletal myogenesis is controlled by mTOR and nutrients. J Cell Biol 163, 931-936 (2003); MS Yoon, J. Chen, PLD regulates myoblast differentiation through the mTOR-IGF2 pathway. J Cell Sci 121, 282-289 (2008); A. Polesskaya et al., Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency. Genes Dev 21, 1125-1138 (2007)), the cargo receptors that regulate the biosynthetic transport of newly synthesized IGF2 remain largely unknown.

[0148] We also reconstructed the release of IGF2 into COPII vesicles using an in vitro vesicle formation assay to quantify the efficiency of cargo packaging. Using these approaches, we identified that the p24 family protein TMED10 acts as a cargo receptor to mediate ER export of IGF2 for myoblast differentiation. Furthermore, we found that TMED10 regulates ER export of sortilin and that sortilin is important for TGN export of IGF2. These studies reveal a molecular mechanism that regulates IGF2 secretion along the secretory pathway for muscle stem cell differentiation.

[0149] We performed co-immunoprecipitation (co-IP) experiments to reveal proteins that interact with the HA-tagged N-terminal fragment of sonic hedgehog (ShhN-HA) or HA-tagged IGF2 (IGF2-HA) (see X. Tang et al., ASURF4-to-proteoglycan relay mechanism that mediates the sorting and secretion of a tagged variant of sonic hedgehog. Proc Natl Acad Sci USA 119, e2113991119 (2022)). Label-free quantitative mass spectrometry analysis of immunoprecipitated proteins identified the transmembrane protein TMED10, which binds more strongly to IGF2-HA than to ShhN-HA (see X. Tang et al., above). TMED10 is an ER-localized and Golgi-localized transmembrane protein belonging to the p24 family. To test whether TMED10 is important for IGF2 secretion, we used the RUSH transport assay (see the above-mentioned literature X. Tang et al.; and literature G. Boncompain et al., Synchronization of secretory protein traffic in populations of cells. Nat Methods 9, 493-498 (2012); Y. Mao et al., The exocyst functions in niche cells topromote germline stem cell differentiation by directly controlling EGFR membrane trafficking. Development 146 (2019)). In this experiment, human IGF2 (aa: 25-180, signal peptide removed) encoding an EGFP tag, a streptavidin binding peptide (SBP) and a C-terminal HA tag was used (referred to as RUSH-IGF2-HA or RUSH-IGF2 25-180 -HA) plasmid to transfect HeLa cells ( Figure 1A ). The plasmid also encodes streptavidin fused to a C-terminal ER retention signal (Lys-Asp-Glu-Leu; Str-KDEL). Due to the binding between streptavidin and SBP, RUSH-IGF2-HA is retained in the ER when expressed ( Figure 1B, 0 min). Note that the RUSH system does not disrupt ER export, but rather creates an imbalance that favors retention over export. Thus, in the absence of biotin, the majority of the cargo is retained in the ER. When cells are incubated with biotin, SBP uncouples from streptavidin, thereby releasing RUSH-IGF2-HA from the ER retention process ( Figure 1B When cells were incubated with biotin for 20 min, more than 80% of the cells showed Golgi-localized IGF2 ( Figure 1B After 120 minutes of biotin treatment, the signal of RUSH-IGF2-HA was greatly reduced ( Figure 1B , indicating that IGF2 is secreted outside the cell or delivered to the lysosome for degradation.

[0150] We then performed siRNA knockdown (KD) experiments to examine whether TMED10 is required for IGF2 secretion. In HeLa cells transfected with siRNA targeting TMED10, the expression of TMED10 was greatly reduced ( Figure 1C ). It is noteworthy that the secretion efficiency of RUSH-IGF2-HA was significantly reduced in TMED10KD cells ( Figure 1D , compare lanes 4 and 8, and Figure 1E ), indicating that TMED10 plays an important role in IGF2 secretion. In contrast, the secretion of another protein, ShhN, was not affected in TMED10 KD cells ( Figure 1F and Figure 1G ), thus demonstrating that TMED10 is a specific regulator of IGF2 rather than a general regulator.

[0151] We then generated TMED10 knockout (KO) HeLa cells to examine the effect of TMED10 deficiency on IGF2 secretion. Western blot analysis showed that TMED10 was completely deleted in TMED10 KO HeLa cells ( Fig. 8A ). In TMED10 KO cells, the efficiency of RUSH-IGF2-HA secretion was also significantly reduced ( Figure 8B , compare lanes 4 and 8, and Figure 8C ), thus demonstrating that TMED10 is essential for IGF2 secretion.

[0152] We noticed that the size of secreted RUSH-IGF2-HA detected by anti-HA antibody in cell lysates was similar to that of RUSH-IGF2-HA, suggesting that the secreted RUSH-IGF2-HA we detected was not the cleaved form. A possible explanation is that we used an antibody that detects the HA tag located at the C-terminus of IGF2 for immunoblotting analysis. We repeated the experiment by using RUSH-IGF2 with an N-terminal HA tag (referred to as RUSH-HA-IGF2) to monitor the secretion of both big IGF2 and mature IGF2. We detected two bands in the culture group, and their molecular weights matched the expected molecular weights of pro-IGF2 and mature IGF2 ( Figure 1H , lane 4). Consistent with previous analyses, secretion of both pro-IGF2 and mature IGF2 was reduced after TMED10 KD ( Figure 1H , compare lanes 4 and 8, and Fig. 1I ).

[0153] We found that the abundance of RUSH-IGF2-HA in cell lysates from TMED10 KD or KO groups was reduced after biotin treatment ( Figure 1H , Lanes 5-6, Figure 8B , lanes 5-6). We hypothesized that some RUSH-IGF2-HA had been degraded rather than secreted. To test this, we performed a RUSH assay using TMED10 KO cells and treated the cells with a lysosomal inhibitor (bafilomycin A1) or a proteasome inhibitor (MG132) in the presence of biotin. We found that bafilomycin A1 treatment increased the abundance of RUSH-IGF2-HA in biotin-treated cell lysates, whereas MG132 treatment showed only a modest effect ( Fig.8D ), indicating that the decreased RUSH-IGF2-HA protein level in cell lysates after biotin treatment was mainly due to lysosomal degradation.

[0154] Example 2—TMED10 is important for IGF2 packaging into COPII vesicles

[0155] Next, we analyzed which step of IGF2 secretion TMED10 is involved in. We found that knockdown of TMED10 caused defects in ER-to-Golgi trafficking of RUSH-IGF2-HA ( Figure 2A and Figure 2B ,as well as Figure 2C This defect was also observed in TMED10 KO cells ( Fig. 9A ,as well as Fig. 9BThis defect was rescued by expression of TMED10-FLAG in TMED10 KO cells ( Fig. 9C ,as well as Fig.9D ), indicating that TMED10 is important for the ER-to-Golgi trafficking of RUSH-IGF2-HA. When TMED10-FLAG was co-expressed with RUSH-IGF2-HA in the absence of biotin, it localized to the ER in most TMED10- and IGF2-co-expressing cells ( Fig. 10A ; Panels AC). In this case, only about 4% of the co-expressing cells showed detectable TMED10-FLAG localization in the juxtanuclear region ( Fig. 10B ). After 10 minutes of biotin treatment, RUSH-IGF2-HA was located in the juxtanuclear Golgi region ( Fig. 10A ; Panels D and F). In this case, TMED10-FLAG colocalized with RUSH-IGF2-HA in the juxtanuclear Golgi region in ∼60% of the co-expressing cells ( Fig. 10A ; Images D and F, and Fig. 10B The percentage of cells showing co-expression of juxtanuclear TMED10-FLAG in the presence of biotin was significantly higher than that detected in the absence of biotin ( Fig. 10B ). These analyses indicate that ER retention of IGF2 causes accumulation of TMED10 at the ER. These analyses also indicate that TMED10 is transported from the ER to the Golgi along with RUSH-IGF2-HA. We then performed similar analyses in cells co-expressing a RUSH construct of ShhN (RUSH-ShhN-HA) and TMED10-FLAG ( Fig. 10C and Fig. 10D Our findings revealed that TMED10-FLAG was not co-transported with RUSH-ShhN-HA from the ER to the Golgi after biotin treatment ( Fig. 10D ). These analyses indicate that TMED10 co-translocates with IGF2, but not with ShhN, from the ER to the Golgi.

[0156] We assume that TMED10 plays the role of cargo receptor to regulate IGF2 packaging into COPII vesicles. To test this hypothesis, we used vesicle formation assay to reconstruct the process of IGF2 release into COPII vesicles (see reference L.Niu et al., Atlastin-mediated membrane tethering is critical for cargo mobility and exit from the endoplasmic reticulum. Proc Natl Acad Sci USA 116, 14029-14038 (2019); Y.Huang et al., An in vitro vesicle formation assay reveals cargoclients and factors that mediate vesicular trafficking. Proc Natl Acad Sci U SA 118 (2021)). HEK293T cells transfected with RUSH-IGF2-HA were permeabilized by digitonin. After permeabilization, semi-intact cells were washed with cold KOAc buffer to remove cytoplasmic proteins. Semi-intact cells were then incubated with a GTP and ATP regeneration system (ATPrS) at 32°C in the presence or absence of biotin, rat liver cytosol (RLC), and a GTPase-deficient mutant of SAR1A, SAR1A(H79G). Figure 2D The released vesicles after incubation were then isolated by centrifugation and analyzed by immunoblotting ( Figure 2D When the vesicle formation assay was performed in the presence of RLC, RUSH-IGF2-HA was detected in the vesicle fraction ( Figure 2E , lane 3). We detected biotin-independent budding of RUSH-IGF2-HA, indicating that the RUSH system does not block ER export. When the assay was performed in the presence of biotin, the abundance of IGF2 in the vesicle fraction was increased, which may be due to release from the retention process ( Figure 2E , compare lanes 3 and 4). Addition of SAR1A(H79G) blocked the vesicular release of RUSH-IGF2-HA ( Figure 2E , compare lanes 4 and 5). These results indicate that the assay successfully reconstituted the release of IGF2 into COPII vesicles. Notably, in both conditions (with or without biotin, Figure 2F and Figure 2G), knockdown of TMED10 caused a significant decrease in the abundance of RUSH-IGF2-HA in transport vesicles, indicating that TMED10 is important for the packaging of RUSH-IGF2 into COPII vesicles.

[0157] In yeast, ERV25 (yeast homolog of human TMED10) forms a heteromeric complex with EMP24, ERP1 and ERP2 (yeast homologs of human TMED2, 4 and 7, respectively) (see M. Marzioch et al., Erp1p and Erp2p, partners for Emp24pand Erv25p in a yeast p24 complex. Mol Biol Cell 10, 1923-1938 (1999)). Their protein levels are interdependent, and these proteins act in a synergistic manner (see M. Marzioch et al., above). In mammals, TMED10 exists in hetero-oligomeric complexes with TMED2, TMED7 and TMED9 (see D. Gommel et al., p24 and p23, the major transmembrane proteins of COPI-coated transport vesicles, form hetero-oligomeric complexes and cycle between the organelles of the early secretory pathway. FEBS Lett 447, 179-185 (1999); J. Fullekrug et al., Localization and recycling of gp27 (hp24gamma3): complex formation with other p24 family members. Mol Biol Cell 10, 1939-1955 (1999)). Then, we analyzed the budding efficiency of two p24 family proteins TMED2 and TMED7, as well as another cargo receptor ERGIC53 in control cells and TMED10 knockdown cells. Consistent with previous reports, the abundance of TMED7 and TMED2 in cell lysates was significantly reduced in TMED10 KD cells ( Figure 2H ), thus indicating a mutual dependence between TMED family proteins to support their stability. As a result, the abundance of these two cargo proteins in the vesicle fraction was also reduced ( Figure 2H Interestingly, we found that the packaging efficiency of ERGIC53 was increased in TMED10 KD cells ( Figure 2Hand Fig.2I ).

[0158] Next, we analyzed the colocalization between IGF2 and TMED10 using a digitonin permeabilized cell assay. We have previously demonstrated that this assay locks the ER export process at the sorting step (see X. Tang et al., ASURF4-to-proteoglycan relay mechanism that mediates the sorting and secretion of a tagged variant of sonic hedgehog. Proc Natl Acad Sci USA 119, e2113991119 (2022)), which provides a convenient method to analyze the colocalization between cargo receptors and cargo molecules. Cells co-expressing RUSH-IGF2-HA and TMED10-FLAG were permeabilized by digitonin and washed with high salt buffer to remove endogenous cytoplasmic proteins. The cells were then incubated with rat liver cytosol, biotin and GTPγS for 15 minutes. After incubation, RUSH-IGF2-HA and TMED10-FLAG showed a punctate localization pattern ( Fig.11A Many IGF2 puncta overlap with TMED10 puncta ( Fig.11A , Fig. 11B and Fig. 11C ). This analysis showed that IGF2 colocalizes with TMED10 upon exiting the ER.

[0159] Example 3—Residues 112-140 of IGF2 are important for ER-to-Golgi trafficking of IGF2

[0160] Next, we addressed the question of which motif of IGF2 is the major determinant of IGF2 trafficking and secretion. We performed sequence alignments of different IGF2 homologous gene sequences and synthesized different IGF2 truncated proteins; each containing one or several different highly conserved regions ( Figure 3A ). Using the RUSH assay, we examined the ER-to-Golgi trafficking of these mutant constructs. Interestingly, we found that RUSH-IGF2 was expressed in more than 90% of cells after 10 min of biotin treatment. 25-48 -HA is located in the ER ( Figure 3B and Figure 3C ). RUSH-IGF2 25-97 -HA also showed defects in ER-to-Golgi trafficking ( Figure 3B ; Image D and Figure 3CIn contrast, RUSH-IGF2 was expressed in approximately 80% of cells after 10 min of biotin treatment. 98-180 -HA localizes to the Golgi apparatus ( Figure 3B ; Image C and Figure 3C ), which indicates that IGF2 98-180 It is a key part in the transport of IGF2 from the ER to the Golgi apparatus.

[0161] Sequence alignment showed that residues between positions 112 and 140 of IGF2 are conserved among species ( Figure 3A , highlighted in green boxes). To test whether these residues are important for ER export of IGF2, we generated IGF2 lacking these residues. 98-180 RUSH constructs (RUSH-IGF2 98-180、Δ112-140 -HA). After 10 or 20 minutes of biotin treatment, RUSH-IGF2 in most cells 98-180 -HA shows juxtanuclear localization ( Figure 3B ; Image GO and Figure 3D ). In contrast, most expressed RUSH-IGF2 98-180、Δ112-140 -HA cells showed ER form ( Figure 3B ;Picture PX and Figure 3D ). Further analysis showed that residues 112-140 in IGF2 were sufficient to deliver SBP-EGFP from the ER to the Golgi with an efficiency similar to that of full-length IGF2 ( Fig. 12A and Fig. 12B Together, these observations suggest that IGF2 112-140 It is the ER-to-Golgi transport motif of IGF2.

[0162] Example 4—Interaction of IGF2 with the gold domain of TMED10 is dependent on residues 112-140 in IF2

[0163] We then performed co-immunoprecipitation (co-IP) experiments using HEK293T cells co-transfected with plasmids encoding FLAG-tagged TMED10 (TMED10-FLAG) and HA-tagged IGF2 or ShhN (IGF2-HA or ShhN-HA). In the co-IP assay, the cross-linker DSP was used to stabilize the interaction. The co-IP assay showed that IGF2-HA bound to TMED10-FLAG ( Figure 4A The percentage of IGF2-HA bound to TMED10-FLAG was significantly higher than the percentage of ShhN-HA bound to TMED10 in cell lysates ( Figure 4A and Figure 4B), indicating that TMED10 specifically interacts with IGF2.

[0164] TMED family proteins have a highly conserved structure. The lumen of TMED protein consists of a signal sequence (SS), a Golgi dynamics (GOLD) domain, and a coiled coil (CC) region (see N. Pastor-Cantizano, JC Montesinos, C. Bernat-Silvestre, MJ Marcote, F. Aniento, p24 family proteins: key players in the regulation of trafficking along the secretory pathway. Protoplasma 253, 967-985 (2016); M. Zhang et al., A Translocation Pathway for Vesicle-Mediated Unconventional Protein Secretion. Cell 181, 637-652 e615 (2020)) ( Figure 4C). The cytoplasmic part of many TMED proteins includes two C-terminal hydrophobic residues that promote ER export (see document N.Nakamura et al., Identification of potential regulatory elements for the transport of Emp24p. Mol Biol Cell 9, 3493-3503 (1998)) and a dilysine motif (KK) important for ER retention (see document R.Aber, W.Chan, S.Mugisha, LAJerome-Majewska, Transmembraneemp24 domain proteins in development and disease.Genet Res (Camb) 101, e14 (2019)). Although TMED10 has a dilysine motif in its cytoplasmic domain, it lacks hydrophobic residues at its C-terminus. Since TMED10 forms a complex with other proteins of the TMED10 family, TMED10 can be enriched in COPII vesicles through the ER export motifs present in other members of the TMED family. The GOLD domain is involved in the recognition of cargo proteins (see the literature V. Anantharaman, L. Aravind, The GOLD domain, a novel protein module involved in Golgi function and secretion. Genome Biol 3, research 0023 (2002)). Then, we generated TMED10 with a FLAG tag. 1-130 , which includes an SS motif and a GOLD domain, to test whether the GOLD domain is sufficient to support the interaction. 1-130 The abundance of IGF2-HA bound to -FLAG was significantly higher than that bound to full-length TMED10-FLAG ( Figure 4C , compare lanes 4 and 5, and Figure 4D ), indicating that TMED10 binds IGF2 via its lumenal GOLD domain.

[0165] Since ER-to-Golgi trafficking of IGF2 depends on residues 112 and 140, we next examined whether this motif is important for the IGF2-TMED10 interaction. We found that deletion of this motif significantly reduced the abundance of IGF2-HA bound to TMED10-FLAG ( Figure 4E , compare lanes 3 and 4, and Figure 4FWe then performed peptide binding assays to investigate whether this interaction was direct. A synthetic peptide corresponding to residues 112-140 of IGF2 (IGF2 112-140 ) were covalently linked to the beads. The beads were then incubated with purified GST or GST-tagged TMED10 GOLD domain (GST-TMED10 1-130 ) were incubated together. The results showed that 112-140 Binding of GST-TMED10 1-130 The abundance of GST was significantly higher than that of peptide-bound GST ( Figure 4G and Figure 4H ), indicating that the ER export motif of IGF2 directly interacts with the GOLD domain of TMED10.

[0166] Example 5—TMED10 is important for IGF2 secretion by C2C12 cells for muscle stem cell differentiation

[0167] We then examined whether TMED10 is important for the secretion of IGF2 by mouse C2C12 myoblasts. We collected culture medium from incubated C2C12 cells transfected with control siRNA or siRNA against TMED10. Proteins in the culture medium were subjected to TCA precipitation and then analyzed by label-free quantitative mass spectrometry to compare the abundance of proteins detected in the culture medium from the two experimental groups. In two replicates, the abundance of IGF2 in the culture medium of TMED10 knockdown cells was greatly reduced compared with that of control cells. In addition to IGF2, we identified 52 secreted proteins whose abundance in the culture medium of control cells was at least 1.9-fold higher than that detected in the culture medium of TMED10 knockdown cells in each replicate. When compared with control cells incubated under the same conditions, the expression of the myoblast differentiation marker myogenin was significantly reduced in TMED10 knockdown cells incubated with differentiation medium (DM) ( Figure 5A Addition of purified IGF2 to the differentiation medium rescued myogenin expression in TMED10 knockdown cells ( Figure 5A ,as well as Figure 5B Quantitative results in ( ). These results suggest that TMED10 plays an important role in myoblast differentiation in the following way: TMED10 acts as a cargo receptor to enrich IGF2 into COPII vesicles, which is a key process for IGF2 secretion. To further analyze the salvage effect, we analyzed myotube formation in C2C12 cells by staining myosin heavy chain (MHC), a marker protein of myotubes. Consistent with our western blot results, myotubes became shorter and thinner after knockdown of TMED10 ( Figure 5CAfter incubation with purified IGF2, myotube formation was rescued and the myotubes became longer and thicker ( Figure 5C ). We then quantified the differentiation index after the C2C12 differentiation assay. In TMED10 KD cells, the differentiation index was significantly reduced, and this defect was rescued by purified IGF2 ( Figure 5D ). These analyses suggest that TMED10 regulates C2C12 differentiation in an autocrine manner. In conclusion, we reveal that TMED10 is important for IGF2 packaging into COPII vesicles for delivery of IGF2 from the ER to the Golgi apparatus, and that this step is critical for myoblast differentiation.

[0168] Example 6—sortilin is another cargo customer of TMED10

[0169] Next, we sought to identify other cargo proteins that depend on TMED10 for enrichment into transport vesicles. We have previously developed a vesicle formation assay combined with a label-free quantitative mass spectrometry method that revealed cargo clients of two ER cargo receptors, ERGIC53 and SURF4 (see L. Niu et al., supra). We used a similar approach to discover cargo clients of TMED10. Large-scale vesicle formation assays were performed using donor membranes provided by wild-type (WT) or TMED10 KO HeLa cells. Label-free quantitative mass spectrometry analysis was then performed to compare protein sequence analysis of vesicles generated by these two experimental groups (WT group and TMED10 KO group). We detected peptides matching TMED10 in vesicle fractions generated by TMED10 KO cells. One possible explanation is that negligible amounts of TMED10 may continue to exist in rat liver cytosol prepared from rat liver, and these TMED10 may be associated with vesicles after the vesicle formation assay. Although this residual amount of protein cannot be detected by immunoblotting, it can be detected using mass spectrometry with the ability to detect low picogram range proteins. We found that based on two biological replicates, the abundance of a series of transmembrane proteins in the vesicle fractions of the TMED10 KO group was significantly reduced ( Fig. 6A , the average fold change of TMED10KO / WT was <0.5). These identified transmembrane proteins include several p24 family proteins: TMED1, TMED2, TMED3, TMED4, TMED5, TMED7, and TMED9 ( Fig. 6A This decrease may be caused by TMED10 depletion inducing the degradation of these TMED proteins, thereby reducing their presence not only within cells but also in vesicle fractions.

[0170] Furthermore, we identified a Golgi-localized and plasma membrane-localized transmembrane protein, sortilin, that depends on TMED10 for its enrichment into transport vesicles ( Fig. 6A , highlighted in red, Figure 6B ). Sortilin is a single-pass transmembrane protein belonging to the vacuolar protein sorting 10 protein (Vps10p) family (see S. Ouyang, B. Jia, W. Xie, J. Yang, Y. Lv, Mechanism underlying the regulation of sortilin expression and its trafficking function. J Cell Physiol 235, 8958-8971 (2020)). Here, we found that TMED10 acts as a cargo receptor that mediates the ER-to-Golgi transport of sortilin.

[0171] The total level of sortilin in both cell lysates and vesicle fractions of the KO group was significantly reduced ( Figure 6B To investigate whether TMED10 is important for the ER-to-Golgi trafficking of newly synthesized sortilin, we generated RUSH constructs of sortilin and performed RUSH transport assays in WT or TMED10 KO HeLa cells. We found that ER-to-Golgi trafficking of sortilin was severely impaired in KO cells ( Figure 6C and Fig.6D Indeed, after 40 min of biotin treatment, most of the sortilin was trapped at the ER. This defect was rescued by transfection of TMED10-FLAG in KO cells ( Figure 6C and Fig.6D These results suggest that TMED10 is also required for ER-to-Golgi trafficking of sortilin. We then performed a vesicle formation assay and immunoisolated vesicles enriched with TMED10-HA ( Fig. 6E Our findings revealed that these isolated vesicles contain RUSH-sortilin-Myc and TMED2 ( Fig. 6F and Figure 6G ), but not SURF4 and ERGIC53 ( FIG. 6F to FIG. 6G ). This analysis showed that sortilin and TMED2 were present in the same vesicles as TMED10, unlike SURF4 and ERGIC53.

[0172] Example 7—Sortilin regulates TGN export of IGF2

[0173] Our previous analysis showed that ER-to-Golgi transport of IGF2 and sortilin is mediated by TMED10. Sortilin mediates insulin-dependent glucose transport in myocytes and is essential for myogenesis (M. Ariga, T. Nedachi, H. Katagiri, M. Kanzaki, Functional role of sortilin in myogenesis and development of insulin-responsive glucose transport system in C2C12 myocytes. J Biol Chem 283, 10208-10220 (2008); G. Huang et al., Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin. Mol Biol Cell 24, 3115-3122 (2013)). Interestingly, we found that knockdown of sortilin significantly reduced the efficiency of RUSH-HA-IGF2 secretion after biotin treatment ( FIG. 7A to FIG. 7C ), indicating that sortilin plays a key role in IGF2 secretion. To determine whether sortilin is required for ER-to-Golgi transport or TGN-to-PM transport of IGF2, we analyzed RUSH-HA-IGF2 transport in sortilin KD HeLa cells at different time points after biotin treatment. After 20 minutes of biotin treatment, the percentage of cells showing juxtanuclear localization of RUSH-HA-IGF2 was similar in the blank control group and sortilin KD group ( Fig.7D and Fig. 7E ), indicating that knockdown of sortilin does not affect the ER-to-Golgi transport of RUSH-HA-IGF2. After 30 minutes of biotin treatment, RUSH-HA-IGF2 showed punctate structures at the cell periphery in more than 60% of the cells. We hypothesize that these punctate structures are RUSH-HA-IGF2-enriched vesicles derived from the TGN. The percentage of cells showing RUSH-HA-IGF2 punctate structures was significantly reduced in the sortilin KD group compared with the control group ( Fig.7D and Figure 7F These analyses indicate that sortilin is important for TGN export of IGF2 but not for ER export of IGF2.

[0174] We found that two transmembrane proteins (TMED10 and sortilin) ​​cooperate to mediate the secretion of IGF2 along the secretory pathway. Based on our studies, we propose that the secretion of IGF2 is achieved through the following steps: Figure 7G ). First, through IGF2 112-140 Due to the direct interaction between the motif and the TMED10GOLD domain, correctly folded ER-localized pro-IGF2 is captured into COPII vesicles. Next, the vesicles containing pro-IGF2 and TMED10 are delivered to the Golgi apparatus, where pro-IGF2 undergoes O-glycosylation modification and cleavage. At the Golgi apparatus, TMED10 dissociates from IGF2 and recycles to the ER via COPI vesicles. In addition, TMED10 also mediates the ER export of sortilin. After reaching the TGN, sortilin regulates the delivery of IGF2 from the TGN to the cell surface ( Figure 7G ). Therefore, sortilin is important for the post-Golgi trafficking of IGF2. These findings support a key role for TMED10 in indirectly mediating the TGN export of IGF2 by regulating the ER-to-Golgi trafficking of sortilin.

[0175] discuss

[0176] TMED10 is a member of the p24 family. Null mutations in TMED10 cause early embryonic lethality in mice (A. Denzel et al., The p24 family member p23 is required for early embryonic development. Curr Biol 10, 55-58 (2000)).

[0177] Inactivation of one allele of TMED10 in mice causes expansion of flat vesicles of the Golgi apparatus (see the above-mentioned document A. Denzel et al.), and knockdown of TMED9 in HeLa cells causes dispersion of the Golgi apparatus (see the document S. Mitrovic, H. Ben-Tekaya, E. Koegler, J. Gruenberg, HP Hauri, The cargo receptors Surf4, endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-53, and p25 are required to maintain the architecture of ERGIC and Golgi. Mol Biol Cell 19, 1976-1990 (2008)). TMED10 negatively regulates autophagy, and the expression of TMED10 is reduced in patients with Alzheimer's disease (see JHShin et al., Down-regulated TMED10 in Alzheimer disease induces autophagy via ATG4B activation. Autophagy 15, 1495-1505 (2019)).

[0178] The yeast homolog of TMED10 forms a complex with the yeast homolog of TMED2, which regulates the transport of the GPI-anchored protein Gas1p from the ER to the Golgi apparatus (see WJ Belden, C. Barlowe, Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport. J Biol Chem 271, 26939-26946 (1996)). In mammalian cells, TMED10 has also been shown to regulate the surface delivery of GPI-anchored proteins (see S. Takida, Y. Maeda, T. Kinoshita, Mammalian GPI-anchored proteins require p24 proteins for their efficient transport from the ER to the plasma membrane. Biochem J 409, 555-562 (2008)). The GTP-bound form of Rab21 has been shown to interact with TMED10 and regulate the localization of TMED10 at the Golgi apparatus (see T. Del Olmo et al., RAB21 interacts with TMED10 and modulates its localization and abundance. Biol Open 8 (2019)). Immunoprecipitation results showed that both TMED10 and TMED2 showed a preference for interacting with Sec24C and Sec24D, indicating that Sec24C and Sec24D are two Sec24 subtypes involved in TMED10-mediated protein transport at the ER (see C. Bonnon, MW Wendeler, JP Paccaud, HP Hauri, Selective export of human GPI-anchored proteins from the endoplasmic reticulum. J Cell Sci 123, 1705-1715 (2010).). Most of the p24 family proteins are mainly located in the lumen side of the organelle membrane.Their asymmetric nature produces a curvature opposite to that required for vesicle budding, thereby changing the physical properties of the membrane (see A. Copic, CF Latham, MA Horlbeck, JG D'Arcangelo, EA Miller, ER cargo properties specify a requirement for COPII coat rigidity mediated by Sec 13p. Science 335, 1359-1362 (2012)). It has been demonstrated that the scaffolding function of the cargo adaptor Lst1p, the yeast homolog of Sec24, and the outer COPII coat Sec13p is essential for counteracting resistance caused by p24 protein and promoting vesicle formation at the ER (see references A. Copic, CF Latham, MA Horlbeck, JG D'Arcangelo, EA Miller, ER cargo properties specify requirements for COPII coat rigidity mediated by Sec13p. Science 335, 1359-1362 (2012); JG D'Arcangelo et al., Traffic of p24 Proteins and COPIICoat Composition Mutually Influence Membrane Scaffolding. Curr Biol 25, 1296-1305 (2015)).

[0179] Upon reaching the Golgi, cargo molecules dissociate from their clients. We have previously demonstrated that proteoglycans compete with SURF4 for interaction with Shh at the Golgi, causing SURF4 to dissociate from its clients (see X. Tang et al., ASURF4-to-proteoglycan relay mechanism that mediates the sorting and secretion of a tagged variant of sonic hedgehog. Proc Natl Acad Sci U SA 119, e2113991119 (2022)). p24 family proteins have been shown to interact with modified GPI-APs to enrich them into COPII vesicles (see literature M.Fujita et al., Sorting of GPI-anchored proteins into ERexit sites by p24 proteins is dependent on remodeled GPI.J Cell Biol 194, 61-75 (2011); G A Castillon et al., The yeast p24 complex regulates GPI-anchored protein transport and quality control by monitoring anchor remodeling. Mol Biol Cell 22, 2924-2936 (2011)). This interaction is pH-dependent, indicating that they may dissociate from each other at the Golgi apparatus due to pH changes (see the above literature M.Fujita et al.). In addition, it has been confirmed that p24 protein can recycle unmodified GPI-anchored proteins that escape from the Golgi apparatus and return them to the ER in the COPI vesicles (see the above literature G A Castillon et al.). This suggests that the p24 protein plays a key role in monitoring anchor modification to ensure accurate transport of GPI-APs (see GACastillon et al., supra). The transmembrane domain of TMED2 (but not TMED10) has been found to interact specifically with sphingomyelin SM18.This interaction promotes efficient reverse COPI-dependent transport and is involved in regulating the balance between the monomeric and oligomeric states of TMED2 (see FX Contreras et al., Molecular recognition of a single sphingolipid species by a protein's transmembrane domain. Nature 481, 525-529 (2012)).

[0180] Interestingly, TMED10 has also been shown to act as a protein channel to mediate unconventional protein secretion (UPS), a group of leaderless proteins including IL1β, IL-1α, HSPB5, Tau, and annexin A1 (see TANguyen, J. Debnath, Unconventional secretion: cargo channeling by TMED10. Cell Res 30, 713-714 (2020)). Unlike the conventional secretion pathway mediated by TMED10, the UPS with TMED10 as a channel requires direct or indirect interaction between the UPS cargo and the C-terminal tail of TMED10 (see M. Zhang et al., A Translocation Pathway for Vesicle-Mediated Unconventional Protein Secretion. Cell 181, 637-652 e615 (2020)). In the unconventional secretion pathway, TMED10 is triggered by the production of UPS cargo to form a higher-order mono-oligomer, which stabilizes the TMED10 protein channel on the ERGIC membrane for UPS protein transport (see the above-mentioned document M. Zhang et al.). If the oligomeric form of TMED10 forms a channel, the pore of the channel will be highly hydrophobic because the transmembrane residues of TMED10 are mainly hydrophobic. This creates an energy barrier for IL1β and other unconventional cargo proteins to pass through. In addition, IL1β and other unconventional secretory proteins are effectively secreted during infection-induced plasma membrane permeabilization (see document CLEvavold, JCKagan, Diverse Control Mechanisms of theInterleukin-1Cytokine Family. Front Cell Dev Biol 10, 910983 (2022)), indicating that these cargo molecules accumulate in large quantities in the cytoplasm of immune cells. Deletion of the GOLD domain abolished TMED10 mono-oligomerization, indicating that the integrity of the GOLD domain is critical for the mono-oligomerization of TMED10 (see the above-mentioned M. Zhang et al.). Direct interaction between IGF2 and TMED10 GOLD domains may interfere with TMED10 mono-oligomerization. In addition, quantitative mass spectrometry analysis showed that TMED1, 2, 3, 7, and 9 showed a significant reduction in TMED10 KO vesicles ( Fig. 6A ). Therefore, we hypothesized that TMED10 and other p24 family proteins formed hetero-oligomers to package IGF2 into COPII vesicles.

[0181] In addition to the selective capture mechanism, swarming is another method for exporting soluble or membrane-associated proteins from the ER (see C. Barlowe, supra). Export via swarming is independent of cargo receptors or export motifs on cargo. Instead, proteins exported via swarming are packaged into COPII vesicles by default. Using the RUSH assay, we found that RUSH-IGF2, excluding 112-140aa, 98-180 -HA exhibits a kinetic delay in ER-to-Golgi transport ( FIG. 3B to FIG. 3D ). In contrast, the SBP-EGFP tag was combined with IGF2 112-140 Motif fusion efficiently delivers SBP-EGFP to the Golgi apparatus ( Fig. 12A : Figure JL), indicating that aggregation is not an efficient method for mediating ER-to-Golgi trafficking of IGF2. Cross-linking and peptide binding experiments reveal the TMED10 GOLD domain and IGF2 112-140 motifs, thereby indicating that TMED10 directly mediates ER export of IGF2.

[0182] In conclusion, our work provides new insights into the molecular mechanisms that mediate the trafficking of IGF2 along the secretory pathway to exert its physiological functions. Dysregulation of IGF2 activity is a candidate risk factor for tumorigenesis and is associated with a variety of conditions, such as Beckwith-Wiedemann syndrome, Russell-Silver syndrome and Doege-Potter syndrome (see references AJPingetal., Genetic linkage of Beckwith-Wiedemann syndrome to 11p15. Am J Hum Genet 44, 720-723 (1989; AY Kalebi, MJ Hale, ML Wong, T. Hoffman, J. Murray, Surgically cured hypoglycemia secondary to pleural solitary fibrous tumour: case report and update review on the Doege-Potter syndrome. J Cardiothorac Surg 4, 45 (2009); C. Gicquel et al., Epimutation of the telomeric imprinting center regionon chromosome 11p15 in Silver-Russell syndrome. Nat Genet 37, 1003-1007 (2005)). The revealed cytokine and protein interactions important for the secretion of IGF2 provide new therapeutic targets to downregulate IGF2 signaling by blocking IGF2 secretion (see T. Li, F. Yang, Y. Heng, Y. Guo, TMED10 mediates the trafficking of insulin-like growth factor 2 along the secretory pathway for myoblast differentiation PNAS 120 (46) e2215285120).

[0183] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that those skilled in the art may conceive of various modifications or changes based on these examples and embodiments, and thus these modifications or changes will be included in the spirit and scope of the present application and the scope of the appended claims. In addition, any element or limitation of any invention or embodiment disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or embodiment thereof, and all such combinations are included within the scope of the present invention, but are not limited thereto.

[0184] Exemplary embodiments

[0185] Embodiment 1. A pharmaceutical composition for inhibiting IGF2 signaling, the composition comprising an inhibitor of IGF2-TMED10 interaction in a cell and one or more pharmaceutical carriers or excipients.

[0186] Embodiment 2. A composition according to embodiment 1, wherein the inhibitor comprises: (a) a human IGF2 polypeptide comprising residues 112-140 of the human IGF2 sequence (SEQ ID NO: 1); (b) a human TMED10 polypeptide comprising residues 1-130 of the human TMED10 sequence; (c) a human TMED10 polypeptide comprising residues 32-132 of the human TMED10 sequence; (d) a small molecule; (e) a covalent inhibitor; (f) an antibody; or (g) a genome editing tool.

[0187] Embodiment 3. A method for treating a subject with abnormal IGF2 signaling, the method comprising: (a) obtaining a pharmaceutical composition comprising an inhibitor of IGF2-TMED10 interaction in a cell and optionally one or more pharmaceutical carriers or excipients; and (b) administering an effective amount of the composition to the subject, wherein the inhibitor comprises: a human IGF2 polypeptide comprising residues 112-140 of the human IGF2 sequence; a human TMED10 polypeptide comprising residues 1-130 of the human TMED10 sequence; a human TMED10 polypeptide comprising residues 32-132 of the human TMED10 sequence; a small molecule; a covalent inhibitor; an antibody; or a genome editing tool.

[0188] Embodiment 4. A method according to any of the preceding embodiments, wherein the subject is a mammal.

[0189] Embodiment 5. The method of embodiment 4, wherein the mammal is a human.

[0190] Embodiment 6. The method according to any of the preceding embodiments, wherein the subject suffers from Beckwith-Wiedemann syndrome, Russell-Silver syndrome, or Deger-Porter syndrome.

[0191] Embodiment 7. The method according to any of the preceding embodiments, wherein the inhibitor blocks the secretion of IGF2 in the cell.

[0192] Embodiment 8. A method for treating cancer in a subject in need thereof, the method comprising: (a) providing a pharmaceutical composition comprising an inhibitor of IGF2-TMED10 interaction in a cell and optionally one or more pharmaceutical carriers or excipients; and (b) administering an effective amount of the pharmaceutical composition to a subject suffering from cancer, wherein the inhibitor comprises: a human IGF2 polypeptide comprising residues 112-140 of the IGF2 sequence; a human TMED10 polypeptide comprising residues 1-130 of the human TMED10 sequence; a human TMED10 polypeptide comprising residues 32-132 of the human TMED10 sequence; a small molecule; a covalent inhibitor; an antibody; or a genome editing tool.

[0193] Embodiment 9. A method according to any of the preceding embodiments, wherein the subject is a mammal.

[0194] Embodiment 10. The method of embodiment 9, wherein the mammal is a human.

[0195] Embodiment 11. The method according to any of the preceding embodiments, wherein the inhibitor blocks the secretion of IGF2 in the cell.

[0196] Embodiment 12. The method according to any of the preceding embodiments, wherein the composition is administered to the subject by injection.

[0197] Embodiment 13. A method according to any of the preceding embodiments, wherein the inhibitor comprises a CRISPR / CAS9 (RNA-guided targeting) genome editing tool, wherein the CRISPR / CAS9 genome editing tool is used to mutate the IGF2 sequence encoding residues 112-140 of IGF2.

[0198] Embodiment 14. A method according to any of the preceding embodiments, wherein the inhibitor comprises a CRISPR / CAS9 (RNA-guided targeting) genome editing tool, wherein the CRISPR / CAS9 genome editing tool is used to mutate the human TMED10 sequence encoding residues 1-130 of the human TMED10 sequence, wherein the mutated TMED10 blocks the binding of TMED10 to IGF2.

[0199] Embodiment 15. A method according to embodiment 8, wherein the inhibitor comprises a CRISPR / CAS9 (RNA-guided targeting) genome editing tool, wherein the CRISPR / CAS9 genome editing tool is used to mutate the IGF2 sequence encoding residues 112-140 of IGF2.

[0200] Embodiment 16. A method according to embodiment 8, wherein the inhibitor comprises a CRISPR / CAS9 (RNA-guided targeting) genome editing tool, wherein the CRISPR / CAS9 genome editing tool is used to mutate the human TMED10 sequence encoding residues 1-130 of the human TMED10 sequence, wherein the mutated TMED10 blocks the binding of TMED10 to IGF2.

Claims

1. A pharmaceutical composition for inhibiting IGF2 signaling, the composition comprising an inhibitor of IGF2-TMED10 interaction in a cell and one or more pharmaceutical carriers or excipients.

2. The pharmaceutical composition according to claim 1, wherein the inhibitor comprises: (a) a human IGF2 polypeptide comprising residues 112-140 of the human IGF2 sequence [SEQ ID NO: 1]; (b) a human TMED10 polypeptide comprising residues 1-130 of the human TMED10 sequence; (c) a small molecule; (d) a covalent inhibitor; (e) an antibody; or (f) a genome editing tool.

3. A method of treating a subject having aberrant IGF2 signaling, the method comprising: (a) obtaining a pharmaceutical composition comprising an inhibitor of IGF2-TMED10 interaction in a cell and optionally one or more pharmaceutical carriers or excipients; and (b) administering to the subject an effective amount of the composition, wherein the inhibitor comprised by the composition comprises: a human IGF2 polypeptide comprising residues 112-140 of the human IGF2 sequence; A human TMED10 polypeptide comprising residues 1-130 of the human TMED10 sequence; a small molecule; a covalent inhibitor; an antibody or a genome editing tool.

4. The method of claim 3, wherein the subject is a mammal.

5. The method of claim 4, wherein the mammal is a human.

6. The method of claim 3, wherein the subject has Beckwith-Wiedemann syndrome, Russell-Silver syndrome, or Deger-Porter syndrome.

7. The method of claim 3, wherein the inhibitor blocks the secretion of IGF2 in the cell.

8. A method of treating cancer in a subject in need thereof, the method comprising: (a) providing a pharmaceutical composition comprising an inhibitor of IGF2-TMED10 interaction in a cell and optionally one or more pharmaceutical carriers or excipients; and (b) administering an effective amount of the pharmaceutical composition to a subject having cancer, wherein the inhibitor comprises: a human IGF2 polypeptide comprising residues 112-140 of the human IGF2 sequence; A human TMED10 polypeptide comprising residues 1-130 of the human TMED10 sequence; a small molecule; a covalent inhibitor; an antibody; or a genome editing tool.

9. The method of claim 8, wherein the subject is a mammal.

10. The method of claim 9, wherein the mammal is a human.

11. The method of claim 8, wherein the inhibitor blocks the secretion of IGF2 in the cell.

12. The method of claim 8, wherein the composition is administered to the subject by injection.

13. The method of claim 3, wherein the inhibitor comprises a CRISPR / CAS9 (RNA-guided targeting) genome editing tool, wherein the CRISPR / CAS9 genome editing tool is used to mutate the IGF2 sequence encoding residues 112-140 of IGF2.

14. The method of claim 3, wherein the inhibitor comprises a CRISPR / CAS9 (RNA-guided targeting) genome editing tool, wherein the CRISPR / CAS9 genome editing tool is used to mutate a human TMED10 sequence encoding residues 1-130 of the human TMED10 sequence, wherein the mutated TMED10 blocks TMED10 binding to IGF2.

15. The method of claim 8, wherein the inhibitor comprises a CRISPR / CAS9 (RNA-guided targeting) genome editing tool, wherein the CRISPR / CAS9 genome editing tool is used to mutate the IGF2 sequence encoding residues 112-140 of IGF2.

16. The method of claim 8, wherein the inhibitor comprises a CRISPR / CAS9 (RNA-guided targeting) genome editing tool, wherein the CRISPR / CAS9 genome editing tool is used to mutate a human TMED10 sequence encoding residues 1-130 of the human TMED10 sequence, wherein the mutated TMED10 blocks TMED10 binding to IGF2.

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