Method for preventing or treating cancer

Abstract

Provided is a method for preventing or treating cancer in a subject who presents an increased human epidermal growth factor receptor 2 (HER2)-mediated activity, through inhibiting a serine kinase activity of HER2. Also provided is a pharmaceutical composition for use in preventing or treating cancer in a subject in need thereof, wherein the pharmaceutical composition includes an agent having a serine kinase inhibition activity against HER2 and a pharmaceutically acceptable carrier thereof.

Description

[0001]CMUH-0002PCTUS METHOD FOR PREVENTING OR TREATING CANCER BACKGROUND Technical Field The present disclosure provides methods for preventing or treating cancers, particularly cancers in subjects who present an increased human epidermal growth factor receptor 2 (HER2)-mediated activity. Description of Related Art Cancer has been the leading cause of death worldwide for the past decades, accounting for nearly 10 million deaths annually in recent years and posing enormous risk to human health. Therefore, developing novel cancer therapy or improving the efficacy of current cancer treatment has long since been the prime goal of the medical community. Protein kinases mediate reversible phosphorylation of target proteins and are important enzymes that participate in almost every cellular process of a living organism, such as cell-cycle regulation, cellular signaling, initiation of protein synthesis, cell proliferation, cell differentiation, and apoptosis, and are needed for proper embryonic development. It is now well established that mutation or misregulation of members of this family of enzymes is a cause or consequence of many disease states. Among these, many cancers are known to be associated with mutations or abnormal expression of kinases. HER2, also identified as ERBB2 or NEU, is a receptor tyrosine kinase (RTK) that is found to be overexpressed or activated in about 30% of breast cancer (BC) cases. HER2 overexpression has also been found in a variety of other solid tumors, including gastric, liver, ovarian, pancreatic, and colorectal cancers, with various incidences. Patients with increased expression or activation of HER2 (HER2+) showed poorer survival and higher relapse rates compared to other breast cancer subtypes, making HER2+ cancers more aggressive than others. Many therapeutics have been developed to target HER2. As the standard first-line treatments, monoclonal antibodies targeting against HER2 including trastuzumab (Herceptin) and pertuzumab (Perjeta) significantly improve the overall survival of metastatic HER2+ breast cancer. However, trastuzumab only benefits nearly 50% of patients with HER2+ diseases, and resistance to the drug rapidly develops even though CMUH-0002PCTUS the HER2 protein level remains abundant. As a standard second-line option, HER2-targeted antibody-drug conjugates (ADCs), for example, trastuzumab emtansine (T-DM1, Kadcyla), have proven effective in HER2+ metastatic breast cancer after failure of prior trastuzumab therapy. However, nearly 50% of patients are still refractory to T-DM1, and initial responders inevitably develop acquired resistance to T-DM1. HER2 tyrosine kinase inhibitors (TKIs) are used as the third-line therapy; however, the efficacy of HER2 TKIs, including lapatinib (Tykerb) and tucatinib (Tukysa), is short-lived, with median progression-free survival (PFS) of less than one year, especially for trastuzumab-recurrent breast cancer. While HER2-targeted therapies have shown benefits for breast cancer patients, the developments of resistance to these agents, including trastuzumab and lapatinib, as well as the occurrence of brain metastasis, remain major unsolved clinical issues for HER2+ cancers. Hence, there remains an unmet need for an effective treatment of HER2+ cancers. SUMMARY The present disclosure provides a method for preventing or treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a composition comprising an agent having a serine kinase inhibition activity against human epidermal growth factor receptor 2 (HER2) and a pharmaceutically acceptable carrier thereof. In at least one embodiment of the present disclosure, the serine kinase inhibition activity is against at least one of serine 1051 and serine 1054 of HER2. In at least one embodiment of the present disclosure, the agent further has a tyrosine kinase inhibition activity against HER2. In at least one embodiment, the method of the present disclosure prevents or treats cancer in a subject having increased expression of HER2. In at least one embodiment, the method of the present disclosure prevents or treats cancer in a subject having increased activation of HER2. In at least one embodiment of the present disclosure, the subject is previously treated with an anti-cancer therapeutic agent. In at least one embodiment of the present disclosure, the subject develops resistance to the anti-cancer therapeutic agent. In at least one embodiment of the present disclosure, the anti-cancer therapeutic agent is at least one of trastuzumab, pertuzumab, trastuzumab emtansine, trastuzumab deruxtecan, lapatinib, neratinib, pyrotinib, and tucatinib. In at least one embodiment of the present disclosure, the subject has CMUH-0002PCTUS refractory cancer. In at least one embodiment, the cancer prevented or treated by the method of the present disclosure is colorectal cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, head and neck cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), glioblastoma, bladder cancer, or cervical cancer. In at least one embodiment, the cancer prevented or treated by the method of the present disclosure is gastric cancer or breast cancer. In at least one embodiment of the present disclosure, the cancer has a brain metastasis. In at least one embodiment of the present disclosure, the agent administered to said subject has a structure of with a simplified molecular-input line-entry system (SMILES) of CCN3CCCC(NC2CC(C)NC(NC(=N)NC1CCC(CL)C(CL)C1)N2)C3. In at least one embodiment of the present disclosure, the agent administered to said subject is 1-(3,4-dichlorophenyl)-2-{4-[(1-ethyl-3- piperidinyl)amino]-6-methyl-2-pyrimidinyl}guanidine. In at least one embodiment of the present disclosure, the agent administered to said subject has a structure of CMUH-0002PCTUS with a SMILES of CCN(CC)CC3CCC(NC2CC(C)NC(NC(=N)NC1CCC(CL)C(CL)C1)N2)CC3. In at least one embodiment of the present disclosure, the agent administered to said subject is 1-(3,4-dichlorophenyl)-2- [4-({4-[(diethylamino)methyl]phenyl}amino)-6-methyl-2-pyrimidinyl]guanidine. In at least one embodiment of the present disclosure, the agent administered to said subject has a structure of with a SMILES of ClC(C=C1)=C(Cl)C=C1NC(=N)NC(=NC(=C1)NCC(CC2)CN2CC)N=C1C. In at least one embodiment of the present disclosure, the agent administered to said subject is 1-(3,4-dichlorophenyl)-2-(4- {[(1-ethyl-3-pyrrolidinyl)methyl]amino}-6-methyl-2-pyrimidinyl)guanidine. The present disclosure also provides a method for preventing or treating cancer in a subject in need thereof, comprising inhibiting a serine kinase activity of HER2. In at least one embodiment of the present disclosure, at least one of serine 1051 and serine 1054 of the serine kinase activity of HER2 is inhibited. In at least one embodiment, the method of the present disclosure further comprises inhibiting a tyrosine kinase activity of HER2. The present disclosure further provides a method for identifying an agent for preventing or treating cancer in a subject in need thereof, comprising screening for a compound having a kinase inhibition activity against at least one of serine 1051 and serine 1054 of HER2. In at least one embodiment, the method of the present disclosure further comprises screening for a compound having a kinase inhibition activity against a tyrosine of HER2. In at least one embodiment of the present disclosure, the tyrosine is at least one of Tyr877, Tyr1005, Tyr1023, Tyr1112, Tyr1139, Tyr1196, Tyr1221, Tyr1222, and Tyr1248 of HER2. The present disclosure also provides a method for preventing or treating cancer in a subject in need thereof, comprising CMUH-0002PCTUS administering to said subject a therapeutically effective amount of the agent identified from any of the above methods. The present disclosure further provides a pharmaceutical composition for use in preventing or treating cancer in a subject in need thereof, the pharmaceutical composition comprising an agent having a serine kinase inhibition activity against human epidermal growth factor receptor 2 (HER2) and a pharmaceutically acceptable carrier thereof. In at least one embodiment of the present disclosure, the serine kinase inhibition activity is against at least one of serine 1051 and serine 1054 of HER2. In at least one embodiment of the present disclosure, the pharmaceutical composition further has a tyrosine kinase inhibition activity against HER2. In at least one embodiment of the present disclosure, the subject has increased expression of HER2. In at least one embodiment of the present disclosure, the subject has increased activation of HER2. In at least one embodiment of the present disclosure, the subject is previously treated with an anti-cancer therapeutic agent. In at least one embodiment of the present disclosure, the subject develops resistance to the anti-cancer therapeutic agent. In at least one embodiment of the present disclosure, the anti-cancer therapeutic agent is at least one of trastuzumab, pertuzumab, trastuzumab emtansine, trastuzumab deruxtecan, lapatinib, neratinib, pyrotinib, and tucatinib. In at least one embodiment of the present disclosure, the subject has refractory cancer. In at least one embodiment of the present disclosure, the cancer is colorectal cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, head and neck cancer, pancreatic cancer, non-small cell lung cancer, glioblastoma, bladder cancer, or cervical cancer. In at least one embodiment of the present disclosure, the cancer is gastric cancer or breast cancer. In at least one embodiment of the present disclosure, the cancer has a brain metastasis. In at least one embodiment of the present disclosure, the agent has a structure of CMUH-0002PCTUS The present disclosure further provides a pharmaceutical composition for use in preventing or treating cancer in a subject in need thereof, the pharmaceutical composition comprising an agent identified by screening for a kinase inhibition activity against at least one of serine 1051 and serine 1054 of human epidermal growth factor receptor 2 (HER2). The present disclosure further provides a use of a pharmaceutical composition for manufacture of a medicament for preventing or treating cancer in a subject in need thereof, wherein the pharmaceutical composition comprises an agent having a serine kinase inhibition activity against human epidermal growth factor receptor 2 (HER2) and a pharmaceutically acceptable carrier thereof. In at least one embodiment of the present disclosure, the serine kinase inhibition activity is against at least one of serine 1051 and serine 1054 of HER2. In at least one embodiment of the present disclosure, the agent further has a tyrosine kinase inhibition activity against HER2. BRIEF DESCRIPTION OF THE DRAWINGS The present disclosure can be more fully understood by reading the following detailed descriptions of the examples, with reference made to the accompanying drawings. FIGs.1A to 1I show that HER2 with non-phosphorylated tyrosine still confers the malignant properties of lapatinib (Lap)-resistant cells. FIG.1A shows the schematic of generating a lapatinib-resistant tumor model CMUH-0002PCTUS in HER2 transgenic (Tg) mice; by treating the mice with lapatinib (10 mg / kg daily) orally after surgical removal of primary tumors, tumors that recurred during treatment were defined as lapatinib-resistant. Growth curve of resistant tumors during lapatinib treatment is shown next to the schematic. Immunohistochemical staining of primary and lapatinib-recurrent tumors with indicated markers are also shown (scale bar, 100 µm), where lapatinib-recurrent mouse HER2-Tg breast tumors present high HER2 expression without tyrosine phosphorylations and high proliferative marker Ki67 expression. FIG.1B shows cell viability assay results of HER2-positive BT474 cell lines and its lapatinib-resistant (LR) clone (BT / LR3 clone), as well as SkBr3 and Sk / LR10 cells treated with increasing concentrations of lapatinib for 24 h (n = 3). FIGs. 1C to 1G show the immunoblot analysis of HER2 knockdown efficiency in BT474, BT / LR3, SkBr3 and Sk / LR10 cells (FIG.1C), where FIG.1D shows the cell viability and FIG.1E shows the colony formation.3D spheroid formation with each dot corresponds to a single spheroid (number of spheroid formed: BT474 (pVoid, n = 152; #A, n = 37; #B, n = 38), and BT / LR3 (pVoid, n = 170; #A, n = 14; #B, n = 9)), and the Y-axis indicates spheroid volume (µm3), with unpaired two-tailed Student’s t-test; 3D spheroid formation of HER2-knockdown cells in SkBr3 and Sk / LR10 was assessed by using CellTiter-Glo 3D assay, with paired two-tailed Student’s t-test, n = 6 (FIG. 1F). FIG. 1G shows that migration of parental and LR cell clones were nearly abolished. FIG. 1H shows representative images of human phospho-RTK arrays and corresponding quantification comparing BT474 vs. BT / LR3 and SkBr3 vs. Sk / LR10. FIG. 1I shows that immunoprecipitation of HER2 followed by immunoblotting with a pan-phospho-tyrosine antibody revealed loss of HER2-associated tyrosine kinase activity in LR cells. FIGs. 2A to 2M show identification of HER2 pS1051 / 1054 in lapatinib-resistant cells. FIG. 2A shows that serine phosphorylation detected by anti-phospho-Akt substrate (PAS) antibody in the anti-HER2 immunoprecipitates was increased in response to lapatinib resistance in both BT474 and SkBr3 cells, and FIG. 2B shows the same observation in lapatinib-treated parental BT474 and SkBr3 cells. Total lysates from indicated cell lines treated with or without lapatinib were subjected to IP / WB analysis with indicated antibodies. FIG.2C shows the mass spectrum analysis result in the identification of HER2 pS1054 in BT / LR3 cells. FIG. 2D shows that HER2 S1051 / 1054 are conserved in HER2 among species, and FIG. 2E shows that HER2 CMUH-0002PCTUS S1051 / 1054 are not conserved in other members of EGFR family. FIG.2F shows the dot blot assays performed using phospho-peptides (PP) and non-phospho-peptides (NP) to assess the specificity of pS1051 and pS1054 antibodies. The intensity of the antibody signal was interfered by the corresponding phospho-peptide (P) but not by the non-phospho-peptide (NP). FIG.2G shows the immunoblot analysis of MDA-MB-231 cells stably expressing wild-type HER2, S1051A, S1054A, or S1051A / S1054A (2A) mutants; MSCV: murine stem cell virus. FIG.2H shows immunohistochemical staining of pS1054 in HER2-positive human breast cancer tissues. Peptide competition was performed using either phospho- or non-phospho-S1054 peptides. FIG. 2I shows immunofluorescence staining of HER2 (green) and pS1054 (red) in HEK293T cells transiently transfected with vector, HER2-WT, or HER2-2A and in MDA-MB-231 cells stably expressing the same constructs. FIG. 2J shows immunoblot analysis results of HEK293T cells transfected with HER2, EGFR, HER3, or HER4 constructs probed with pS1051 and pS1054 antibodies. FIGs.2K to 2M show immunoblot analysis of HER2, pTyr1221, pS1051 / 1054, and pErk in the indicated cell lines. In FIG. 2K, HEK293T cells were transfected with HER2 wild-type (WT), phospho-deficient mutant (2A: S1051A / S1054A), phospho-tyrosine-null mutant (9F: Y877F, Y1005F, Y1023F, Y1112F, Y1139F, Y1196F, Y1221F, Y1222F, and Y1248F), or combined 9F2A mutant. In FIGs. 2L and 2M, BT474 cells, SkBr3 cells, and SUM190 cells were treated with lapatinib (+Lap) or tucatinib (+Tu) (1 µM, 24 h) or compared to resistant derivatives: lapatinib-resistant (BT / LR3, Sk / LR10), tucatinib-resistant (BT / TuR1, BT / TuR2), and trastuzumab-resistant (BT / HR). Tubulin serves as a loading control. FIGs. 3A to 3P show that HER2 pS1051 / 1054 correlates with the poor prognosis of HER2-positive (HER2+) and links to HER2+ BC and GC progression. FIG.3A shows the representative immunofluorescence images of matched normal and tumor tissues from HER2-positive breast cancer patients. HER2 (green), pS1054 (red), and nuclei (DAPI, blue) are shown. Quantification of pS1054 signal intensity from 34 matched pairs is presented (right), with paired two-tailed Student’s t-test; scale bar, 100 µm. FIG. 3B shows the immunofluorescence images of gastric tumors and adjacent normal tissues. HER2 (green), pS1054 (red), and DAPI (blue) are shown. Quantification of pS1054 signal intensity in 152 paired tumor and normal tissues is shown in the histogram, with paired two-tailed Student’s t-test; scale bar, 50 µm. FIG. 3C shows a patient- CMUH-0002PCTUS paired vertical mscatterplot showing mean intensity of HER2 pS1054 in nontumor (CK19-) and tumor (CK19+) fractions, **** p < 0.0001. FIG. 3D shows the representative images from Opal multiplex IHC staining of a HER2+ BC tumor tissue. FIG. 3E shows the frequency of CK19+ / HER2+ and CK19+ / pS1054+ in different HER2+ fractions (hi: high; med: medium; lo: low). FIG. 3F shows the representative IHC (top) and immunofluorescence (bottom) images of HER2 pS1054 staining in breast cancer tissues, stratified by staining intensity: negative (-, +) and positive (++, +++, ++++). FIGs. 3G and 3H show the Kaplan-Meier survival analysis of HER2+ breast cancer patients from the CMUH / EDA cohort and gastric cancer patients, respectively, stratified by HER2 pS1054 status. P^value was calculated using log-rank (Mantel-Cox) test. FIG.3I shows the pie charts showing the distribution of pS1054-positive and pS1054-negative cases in primary tumor tissues collected before trastuzumab treatment, among all patients (left), trastuzumab-sensitive (middle), and trastuzumab-resistant cases (right). FIG. 3J shows the association between pS1054 status in pre-treatment primary tumors and response to trastuzumab therapy in HER2-positive breast cancer patients. P value by Pearson’s χ² test. FIG. 3K shows Kaplan-Meier survival analysis of trastuzumab-treated HER2-positive patients from the Xiangya cohort, stratified by pS1054 status. P value by log-rank test. FIG. 3L shows the images of IHC staining for HER2 pS1054, Her2 pY1221 / 2, and HER2 in HER2+ BC patient tumors before and after receiving Herceptin. FIG. 3M shows representative IHC images of HER2 pS1054 staining in metastatic lesions from brain, lung, and liver. Scale bar, 20 µm. FIG. 3N shows the frequency and intensity distribution of HER2 pS1054 positivity across 58 metastatic tumor samples. Pie chart (left) summarizes the percentage of pS1054-positive cases; bar plot (right) shows staining intensity distribution across metastatic sites. IHC scoring: cyan (-), gray (+), purple (++), yellow (+++), red (++++); (-) and (+) are considered pS1054- negative, and (++) to (++++) are pS1054-positive. FIGs. 3O and 3P show that HER2 pS1054 was higher in Herceptin-recurrent brain (FIG. 3O) or lung and liver (FIG. 3P) metastatic tumors than in primary HER2+ breast tumors. FIGs. 4A to 4R show that HER2 pS1051 / 1054 drives cancer stemness and the effects of HER2 S1051 / 1054 mutations in suppressing tumor formation. FIG. 4A shows the representative images and quantification of 3D spheroid volume in BT474 and BT / LR3 cells. Data represent a total of three independent CMUH-0002PCTUS experiments; each dot represented one spheroid (BT474, n = 82; BT / LR3, n = 63). The Y-axis indicated spheroid volume (µm3). Unpaired two-tailed Student’s t-tests; scale bar, 200 µm. FIG. 4B shows confocal immunofluorescence staining of HER2 (green), pS1054 (red), and DAPI (blue) in SkBr3 and Sk / LR10 spheroids. Merged images show colocalization. FIG. 4C shows immunoblot analysis of SkBr3 and Sk / LR10 spheroids for SOX2 and HER2 phosphorylations. FIG. 4D shows representative images and volume quantification of BT / LR3 spheroids when cultured with or without lapatinib (1 µM). Data represent a total of three independent experiments; each dot represented one spheroid (BT / LR3+Lapatinib, n = 61; BT / LR3- Lapatinib, n = 42). The Y-axis indicated spheroid volume (µm3). Unpaired two-tailed Student’s t-tests; scale bar, 200 µm. FIG. 4E shows brightfield and confocal immunofluorescence images of SOX2 (red) and DAPI (blue) in SkBr3 and Sk / LR10 spheroids, with or without lapatinib as indicated. FIG. 4F shows immunoblot analysis of BT / LR3 and Sk / LR10 cells cultured with or without lapatinib (1 µM), probing for SOX2, pS1051, pS1054, pY1221 / 2, HER2, pAkt, and pErk. FIGs. 4G to 4I show gene set enrichment analysis (GSEA) of RNA-seq data from MDA-MB-231 cells stably expressing HER2 wild-type (WT; n = 3 clones) or mutants (S1051A and S1054A; n = 2 clones each). Average expression from S1051A and S1054A was used to represent the mutant group. Expression values were normalized to MSCV vector controls (n = 2 clones). FIG.4G shows the stem cell proliferation gene set; FIG. 4H shows the mammary gland development gene set; and FIG. 4I shows the MUELLER_PLURINET gene sets. FIG.4J shows the heatmap of stemness-related gene expression in MDA-MB-231 cells stably expressing MSCV vector controls (n = 2 clones), HER2 WT (n = 3 clones), or HER2 mutants (n = 2 clones each of S1051A and S1054A). FIG.4K shows immunoblot analysis of stemness markers (SOX2, ALDH1, CD44) in MDA-MB-231 cells expressing HER2 WT, S1051A, S1054A, or double mutant (2A). FIG.4L shows flow cytometry analysis of ALDH+ populations in MDA-MB-231 cells expressing the indicated HER2 constructs. Data shown as mean ± SD (n = 4) with unpaired two-tailed Student’s t-test. FIG.4M shows anchorage-independent growth assessed by soft agar colony formation assays. Representative images and quantification of colony numbers are shown (n = 8); unpaired two-tailed Student’s t-test. FIG.4N shows 3D spheroid formation assays performed in MDA-MB-231 cells. Representative images and spheroid volume quantification are shown from three independent experiments (MSCV, n = 43; WT, n = 98; S1051A, CMUH-0002PCTUS n = 52; S1054A, n = 47; 2A, n = 58). Each dot represents one spheroid; P values by unpaired two-tailed Student’s t-test. FIG.4O shows tumor-free survival curves of HER2 WT or 2A transgenic (Tg) mice with log- rank (Mantel-Cox) test. FIG. 4P shows representative images of tumors (red arrows) in HER2 WT and 2A transgenic (Tg) mice and quantification of tumor numbers per mouse with unpaired two-tailed Student’s t-test. FIG.4Q shows mammary-derived cancer organoids (MDCOs) from HER2 WT and 2A tumors. Representative images (left) and organoid volume quantification (right) from three independent experiments (WT, n = 173; 2A, n = 95) are shown with unpaired two-tailed Student’s t-test. FIG.4R shows IHC analysis of tumors from HER2 WT and 2A mice for SOX2 (stemness), vimentin and E-cadherin (EMT), Ki67 (proliferation), pS1054, pY1221 / 2, and total HER2. FIGs.5A to 5I show that HER2 pS1051 / 1054 promotes brain metastasis. FIG.5A shows the GFP-labeled BT474 and BT / LR3 cells embedded in porcine brain extracellular matrix (B-ECM) and imaged on day 0 and day 7. GFP signal intensity was quantified (n = 3), with unpaired two-tailed Student’s t-test; scale bar, 420 µm. FIG.5B shows that Sk / LR10 clone (labeled with blue 7-amino-4-chloromethylcoumarin (CMAC) dye) grows better in pig brain ECM than in its parental cells. FIG.5C shows higher migration of Sk / LR10 clone evidenced by the increased cell numbers outside of the dome with brain ECM. FIG.5D shows Ingenuity Pathway Analysis (IPA) of RNA-seq data from MDA-MB-231 cells expressing HER2 WT, S1051A, or S1054A, shown as a heatmap of enriched signaling pathways. FIG.5E shows the 2A mutation suppressing the gene set involved in neuron differentiation in GSEA analysis. FIG. 5F shows dissemination assays of MDA-MB-231 cells expressing HER2 WT or 2A, embedded in porcine B-ECM and monitored over 5 days. White dashed circle indicates initial dome boundary (day 0); red dashed lines mark invasive fronts at subsequent time points. FIG. 5G shows quantification of dissemination area over time (n = 30), with two-way repeated-measures ANOVA. FIG.5H shows cell viability in brain ECM at day 5 that was assessed by CellTiter-Glo 3D assay in MDA-MB- 231 cells expressing HER2 WT or 2A (n = 30), with unpaired two-tailed Student’s t-test. FIG. 5I shows that the 2A mutation reduces the brain metastasis of HER2-overexpressing MDA-MB-231 cells injected through internal carotid artery. FIGs.6A to 6I show the validation results of candidate kinases for HER2 pS1051 / 1054 phosphorylation. CMUH-0002PCTUS FIG. 6A shows the comparison of HER2 S1051 / 1054 nearby sequence with the conserved substrate motif of Ser / Thr kinases. FIGs. 6B to 6E show the status of HER2 S1051 / 1054 phosphorylations when treated with inhibitors or gene silencers of Akt, TGFBR1, and PKA. FIG. 6F shows immunoblot analysis of predicted kinases from PhosphoSitePlus, including PHKG1, PKCα, PKCβ, PKCδ, PKCθ, GAK, PBK, BMP2K (BIKE), AAK1, and CAMKII, in BT474, BT / LR3, SkBr3, and Sk / LR10 cells. FIG.6G shows immunoblot analysis of SkBr3 cells treated with pan-PKC inhibitor, sotrastaurin (5 µM, 24h). Phosphorylation levels of HER2 pS1051, pS1054, PKCα / β, and PKCδ / θ were analyzed. FIGs.6H and 6I show siRNA-mediated knockdown of PRKCD (PKCδ) and PBK in Sk / LR10 cells, respectively, followed by immunoblot analysis of pS1051, pS1054, and target knockdown efficiency. FIGs. 7A to 7N show that HER2 possesses a serine kinase activity for HER2 S1051 / 1054 autophosphorylation by using both ATP / GTP as the phosphor-donors. FIG. 7A shows the schematic of the HER2 kinase domain highlighting residues involved in ATP binding and catalysis (left), and FIG. 7A further shows immunoblot analysis (right) of HEK293T cells transfected with HA-tagged HER2 wild-type (WT), kinase-dead mutant (K753M), magnesium-binding-deficient mutant (D863K), or ATP-binding deficient mutant (KGG: K753M / G729D / G732D). FIG.7B shows IVK assays using recombinant HER2 kinase and ATP as the phosphate donor, detected by luminescence (ADP-Glo). Substrates included: synthetic Y peptide (residues 1212-1230), S peptide (residues 1043-1062), and a scrambled negative control (NC). Lapatinib (1 µM) was added where indicated. FIG. 7C shows dot blot analysis of IVK reactions using ATP or GTP as phosphate donors with antibodies against pS1054 and pY1221 / 2. Quantification from three independent experiments is also shown, with P values by unpaired two-tailed Student’s t-test. FIGs.7D and 7E are surface plasmon resonance (SPR) analyses showing non-hydrolyzable nucleotide analogs: nGTP (FIG.7D) and nATP (FIG. 7E), respectively, bound to HER2 kinase domain in nM range affinity. Assays were conducted in the presence of 1 mM MgCl2. The values of equilibrium dissociation constant (KD), maximum binding response (Rmax), and Chi-squared (χ²) were listed individually. RU: resonance unit. FIG. 7F shows the SPR assay showing the binding of nGTP to HER2 in PBS buffer without added MgCl2. FIG. 7G shows purified His- tagged HER2 kinase domain (676-1045) incubated with ATP-, GTP-, or control agarose beads (25 µL). Bound CMUH-0002PCTUS proteins were detected by western blotting using anti-His antibody. FIG. 7H shows lysates from HEK293T cells overexpressing HA-tagged HER2 (1 mg or 2.5 mg total protein) incubated with ATP- or GTP-conjugated agarose beads. ATP beads were used at 2 volumes (2V, 50 µL) and GTP beads at 1 volume (1V, 25 µL). Bound HA-tagged HER2 was detected by immunoblotting. FIG. 7I shows lysates (2.5 mg or 1 mg total protein as indicated) from HA-tagged HER2-overexpressing HEK293T cells treated in vitro with lapatinib at 10, 20, or 40 µM for 4 h (RT) and incubated with ATP-agarose (50 µL), GTP-agarose (25 µL), or resin control (2V). Bound HA-tagged HER2 was detected by immunoblotting. Resin-only conditions were used as negative controls. Individual band intensity was quantified and also shown in each blot. FIG.7J shows that S1051 / 1054- containing peptide shows binding affinity to the purified HER2 kinase domain in the protein thermal shift assay. FIG. 7K shows that HER2 is the serine kinase for pS1051 / 1054 by using both ATP / GTP in in vitro kinase assays. FIG.7L shows molecular docking of GTP / Mg2+(magentas / green) and lapatinib (xyan) to HER2 (pink) by Proteinix Server (AlphaFold 3-based). The residues within 4 Å away from GTP are presented as sticks, confident level ≈ 80. FIGs. 7M and 7N show immunoblot analysis of HEK293T-overexpressing HER2 WT and mutants when treated with indicated lapatinib for 6 h. FIGs. 8A to 8F show that HER2 symmetric dimer drives serine autophosphorylation. FIG. 8A shows schematic representation of HER2 homodimerization types highlighting key mutants broken dimer. FIG. 8B shows immunoblot analysis of HEK293T cells transfected with HA-tagged HER2 WT or dimerization mutants: I714Q (asymmetric), IRVR (EGFR-like symmetric, I748R and V750R), or NRKF (HER3-like symmetric, N764R and K765F). Cells were treated with increasing concentrations of lapatinib (0, 0.1, and 0.5 µM) for 24 h. FIG. 8C shows native PAGE of HA-tagged HER2 WT and mutant-expressing HEK293T cells, with or without lapatinib (0.5 µM, 24 h). HER2 dimers were detected by immunoblotting under long and short exposure. Total and phosphorylated HER2 were detected from SDS-PAGE lysates. FIG.8D shows immunoblot analysis of SkBr3 and T47D stable overexpressing HER2 (T47D / HER2) cells expressing shRNAs targeting EGFR, HER3, or HER4. FIGs. 8E and 8F show in vitro kinase assays using the 2S peptide or the 2Y peptide with increasing concentrations of 2A or 2F peptides. All reactions used ATP as phosphate donor; luminescence signals (relative light unit, RLU) were quantified and expressed as percent change, with P values by unpaired CMUH-0002PCTUS two-tailed Student’s t-test. FIGs. 9A to 9M show the development of HER2 dual-specificity kinase inhibitors. FIG. 9A shows a flowchart of drug screening. FIG. 9B shows the compounds identified by the high-content screening system: immunofluorescence visualization of HER2 pS1054 (red) and HER2 (green) in the upper half panel and the quantitative data of pS1054 / HER2 ratio in the lower half panel. FIG.9C shows the heatmaps of cell viability in HER2-positive parental (Par) and drug-resistant (Res) breast cancer (BC) and gastric cancer (GC) cell lines treated with increasing concentrations of lapatinib, tucatinib, compound PAN-430 or PAN-467 for 24 h. FIG. 9D shows the results of spheroid formation assays performed in 96-well U-bottom ultra-low attachment plates. HER2-positive breast cancer cell lines (SkBr3, Sk / LR10, BT474, and BT / LR3) and gastric cancer cell lines (KATOIII and NCI-N87) were treated with increasing concentrations of compound PAN-430 and PAN-467 for 6 days. Scale bars, 250 µm. FIG. 9E shows the binding affinity of PAN-430 / 467 to the purified HER2 kinase protein determined in SPR analysis. FIG. 9F shows immunoblot analysis of HER2 signaling levels in SkBr3 cells treated with increasing concentrations of compound PAN-430 or PAN-467 for 24 h. FIGs. 9G and 9H show the inhibitory effect of lapatinib, compound PAN-430, or PAN-467 (30 µM) on HER2 pS1054 assessed in IVK assays using recombinant HER2 and GTP as phosphate donor. FIG.9I shows a dual-specificity kinase activity of PAN-467. FIG.9J shows immunofluorescence images of patient-derived cancer organoids (PDCOs) treated with compound PAN-467 for 24 h, stained with propidium iodide (PI, red) and DAPI (blue) to assess cell viability, and HER2 (green) and pS1054 (red) following treatment with compound PAN-467. FIG. 9K shows viability of patient-derived cancer organoids (PDCOs) treated with 10 µM of each compound for the indicated durations (Days 1, 2, 3, 12, and 20), measured by CellTiter-Glo 3D assay. Fold change in viability relative to day 0 is shown. P values were calculated by unpaired two-tailed Student’s t-test. FIG. 9L shows schematic of in vivo treatment study in HER2 transgenic mice following tumor resection. Mice received daily oral gavage of lapatinib or compound PAN-467 (10 mg / kg), and tumor-free survival of HER2 transgenic mice treated with compound PAN-467 or lapatinib (10 mg / kg daily; n = 3 per group). P value was calculated using log-rank (Mantel-Cox) test. FIG. 9M shows tumor volume over time in HER2 transgenic mice treated with lapatinib or compound PAN-467. Data from three mice per group; P values by two-way ANOVA. CMUH-0002PCTUS FIGs. 10A to 10H show the results of molecular docking analysis of HER2 kinase domain for tyrosine / serine phosphorylation. DETAILED DESCRIPTION HER2 plays an oncogenic role in a variety of cancers, including breast and gastric cancers, among several other types of cancers. Through its tyrosine kinase activity, HER2 triggers multiple downstream survival and growth signals for tumor progression. Despite the approval and widespread use of HER2-targeted therapies, including monoclonal antibodies and tyrosine kinase inhibitors (TKIs), insensitivity, acquired resistance, and brain metastasis remain the unsolved challenges. Therefore, the development of an innovative therapeutic strategy aimed at deciphering the intricacies of HER2 is an urgent imperative for addressing the needs of advanced HER2-positive cancer patients. In the present disclosure, a novel GTP-dependent serine kinase activity of HER2 is identified, which regulates serine autophosphorylations independent of tyrosine autophosphorylations. It is noted that HER2 phosphorylations at two specific serine residues, which are enhanced rather than inhibited by current HER2- targeted therapies, mediate drug resistance to HER2-targeted therapies and cancer stemness and are associated with distant metastasis and poor survival rate. This disclosure identifies the dual-specificity kinase (DSK) activity of HER2 and provides HER2-targeted therapeutic agents with a superior anti-cancer activity. It is found that HER2 status remains positive and is tyrosine unphosphorylated in most recurrent and metastatic tumors after failure of HER2-targeted therapy, implying the existence of unknown oncogenic functions of HER2. In this disclosure, the Tyrosine / Serine (Tyr / Ser) dual-specificity kinase activity of HER2 is targeted and inhibited to stop or reverse tumor growth, thereby treating cancer in a subject in need thereof. The following examples are used for illustrating the present disclosure. A person skilled in the art can easily conceive the other effects of the present disclosure, based on the disclosure of the specification. It will be apparent that one or more embodiments may be practiced without specific details. The present disclosure can also be implemented or applied as described in different examples. It is possible to modify or alter the following examples for carrying out this disclosure without contravening its scope for different applications. Titles or subtitles may be used in this disclosure for the convenience of a reader, which shall have no influence CMUH-0002PCTUS on the scope of the present disclosure. In this disclosure, all terms including descriptive or technical terms which are used herein should be construed as having meanings that are obvious to one of ordinary skill in the art. However, the terms may have different meanings according to an intention of one of ordinary skill in the art, case precedents, or the appearance of new technologies. Also, some terms may be arbitrarily selected by the applicant, and in this case, the meaning of the selected terms will be described in detail in the descriptions of the present disclosure. Thus, the terms used herein are defined based on the meaning of the terms together with the descriptions throughout the specification. Throughout the context, the well-known 3 letter and 1 letter codes for the genetically coded amino acids are used. As used in this disclosure, the singular forms “a,” “an,” and “the” include plural referents unless expressly and unequivocally limited to one referent. The term “or” is used interchangeably with the term “and / or” unless the context clearly indicates otherwise. Also, when a part “includes” or “comprises” a component or a step, unless there is a particular description contrary thereto, the part can further include other components or other steps, not excluding the others. As used herein, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B CMUH-0002PCTUS (and optionally including other elements). The phrase “an effective amount” refers to the amount of an active ingredient that is required to result in a reduction, inhibition, or prevention of a disorder or condition, or one or more symptoms of such condition or disorder in a subject. An effective amount will vary, as recognized by those skilled in the art, depending on routes of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment. As used herein, the term “composition” can be prepared according to any method known in the art for the manufacture of pharmaceuticals. Such composition or combination may contain sweetening agents, flavoring agents, coloring agents, and preserving agents. A formulation can be admixed with nontoxic and pharmaceutically acceptable excipients which are suitable for manufacture. Non-limiting formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, lozenges, packets, troches, elixirs, suspensions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterilized injection fluid, packaged powder, on patches, in implants, etc. As used herein, pharmaceutically acceptable carriers, including buffers, are well known in the art and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; physiological saline; sterilized water; isotonic agents; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20thEd. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. As used herein, the term “treat,” “treating,” or “treatment” refers to the application or administration of one or more active agents to a subject afflicted with a disorder, a symptom, or a condition of a disease, or a progression of the disease, with the purpose to cure, heal, relieve, alleviate, alter, remedy, ameliorate, improve, or affect the disorder, the symptom, or the condition of the disease, the disabilities induced by the disease, or the progression of the disease. The term “cancer” refers to a malignant neoplasm. The terms “neoplasm” and “tumor” are used herein CMUH-0002PCTUS interchangeably and refer to an abnormal mass of tissue, wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue. A “neoplasm” or “tumor” may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. A “malignant neoplasm” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm generally has the capacity to metastasize to distant sites. The terms “subject,” “individual,” and “patient,” as used herein, are interchangeable and refer to an animal, especially a mammal. These terms include, but are not limited to, domestic animals, sports animals, primates, and humans. For example, the terms refer to a human. The term “administration” or “administering” used herein refers to introducing, providing, or delivering a pre-determined active ingredient to a subject by any suitable routes to perform its intended function. The following examples are used for illustrating the present disclosure. A person skilled in the art can easily conceive the other effects of the present disclosure, based on the disclosure of the specification. It will be apparent that one or more embodiments may be practiced without specific details. The present disclosure can also be implemented or applied as described in different examples. It is possible to modify or alter the following examples for carrying out this disclosure without contravening its scope for different applications. Titles or subtitles may be used in this disclosure for the convenience of a reader, which shall have no influence on the scope of the present disclosure. EXAMPLES Exemplary embodiments of the present disclosure are further described in the following examples, which should not be construed to limit the scope of the present disclosure. Materials and methods used in this disclosure are shared among the examples and are presented in the following. Cell lines and culture conditions SkBr3 (HTB-30), BT474 (HTB-20), and SUM190 human HER2+ breast cancer (BC) cell lines, BT474- clone 5 (CRL-3247) human HER2+ BC Herceptin-resistant cell line, NCI-N87 (CRL-5822) human HER2+ CMUH-0002PCTUS gastric cancer (GC) cell line, MDA-MB-231 (HTB-26) human triple-negative breast cancer (TNBC) cell line, 293T (CRL-3216) cell line, and L-WRN (CRL-3276) cell line were obtained from American Type Culture Collection (ATCC). SkBr3 and BT474 cell lines were cultured in DMEM / F12 (Dulbecco’s modified Eagle medium / F12) (SH30004.04, HyClone), and BT474-clone 5 and NCI-N87 cell lines were cultured in RPMI-1640 (Roswell Park Memorial Institute-1640) (SH30011.02, HyClone). The lapatinib-resistant (LR) clones were generated from the long-term treatment with lapatinib (GlaxoSmithKline) and maintained in the presence of 1 μM lapatinib during cell culture. MDA-MB-231 cell line was used as retrovirus infection host to generate the stable cell clones with constitutively expressing HER2 wildtype (WT) and mutants, S1051A, S1054A, and 2A. HER2-overexpressing stable clones were maintained in the presence of 2 mg / mL puromycin. The L-WRN cells, used to produce Wnt-3A, R-spondin 3, and noggin-conditioned medium (L-WRN conditioned medium), were cultured in DMEM containing 0.5 mg / mL G-418 and 0.5 mg / mL hygromycin B. All culture media were supplemented with 10% fetal bovine serum (FBS; 10437-028, Gibco) and 1% penicillin-streptomycin (SH40003.01, HyClone). All cells were cultured at 37oC in a humidified atmosphere of 95% air and 5% CO2. Western blotting (WB) Whole cell lysate (WCL) was prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris- HCl, pH 7.4, 1% NP-40, 0.15% sodium deoxycholate (Na-DOC), 150 mM NaCl, and 1 mM ethylenediaminetetraacetic acid (EDTA)) containing protease inhibitors (4693132001, Roche Diagnostics), followed by sonication and centrifugation to collect supernatant. Protein concentration was measured by Bradford assay using Bio-Rad protein assay dye reagent concentrate (5000006, Bio-Rad). The same amount of total proteins was prepared with 1× sample buffer and boiled at 95oC for 5 minutes before loading into sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by WB analysis with indicated proteins. Co-immunoprecipitation and mass spectrum analysis Cells were lysed in the immunoprecipitation buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% NP-40, 1× protease inhibitor cocktail) and prepared as described above for western CMUH-0002PCTUS blotting. For each sample, 1 mg of lysate was incubated with 1 µg of anti-HER2 or mouse IgG antibodies overnight at 4oC under rotation and was then added with 30 μL of protein G for 4 h of rotation at 4oC. Beads were washed 3 times with IP buffer. For phosphatase treatment, protein-bound beads were incubated with lambda protein phosphatase for 30 min at 30oC. Proteins were released by 2× SDS-PAGE loading buffer and analyzed by western blotting. For MS / MS analysis, immunoprecipitated proteins were resolved by SDS-PAGE and visualized by Coomassie staining. Gel slices corresponding to HER2 were excised and subjected to in-gel trypsin digestion. Peptides were analyzed by LC-MS / MS using an electrospray ionization quadrupole time-of-flight (ESI Q-TOF) mass spectrometer. Peak lists were searched using Mascot (Matrix Science) against the UniProt human database. ATP / GTP-conjugated agarose beads pull-down assay The binding affinity of ATP / GTP with HER2 was carried out by pulling down ATP / GTP-conjugated agarose beads as described by Sumita, K. et al. (Mol. Cell (2016) 61, 187-198). Briefly, 1 × 107of HEK293T cells that overexpressed pcDNA6A-HA-HER2 were lysed in the lysis buffer containing 20 mM 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.3), 40 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol (DTT), and protease and phosphatase inhibitor cocktail. Lysates were sheared by repeated passage through a 23G needle and clarified by centrifugation at 20,000 ×g for 30 min at 4oC. The resulting supernatant was incubated with ATP / GTP-conjugated agarose (ab270535 / ab270533, Abcam) for 4 hours at 4oC with gentle rotation. Beads were washed three times with the lysis buffer, and bound proteins were eluted in 3× SDS-PAGE loading buffer for immunoblotting. For recombinant pull-down assays, 2.5 μg of purified His-tagged HER2 protein were incubated with ATP- or GTP-conjugated agarose beads under the same conditions described above. Virus production, infection, and siRNA transfection For short hairpin RNA (shRNA) lentivirus, 2.5 μg shRNA lentiviral plasmid, 0.25 μg pMD.G envelope- expressing plasmid, and 2.25 μg pCMV-D8.91 packaging plasmid were co-transfected into 2.4 × 106HEK293T cells / 6 cm plate by Lipofectamine 2000 transfection reagent (11668019, Invitrogen). Virus-containing media CMUH-0002PCTUS were collected after 48 hours post-transfection. Cells were infected with shRNA lentivirus at the multiplicity of infection (MOI) of 125 and selected with 2 μg / mL puromycin for 3 days before subjected into functional assays. The specific shRNA sequences used are listed in Table 1 below. Table 1. shRNA sequences of the oligonucleotides used For retrovirus production, 15 μg pMSCV retroviral plasmid (HER2-overexpressing plasmid and its mutants) and 15 μg pVSV-G envelope plasmid were co-transfected into 5 × 106GP2-293 cells / 10 cm plate by Xfect transfection reagent (631317, Takara), following the manufacturer’s instructions. MDA-MB-231 cells were infected with HER2-producing retrovirus at the MOI of 3 and selected with 2 μg / mL puromycin for at least 7 days until the colonies were formed. Colonies were sub-cultured in the presence of 1 μg / mL puromycin and checked for a total HER2 expression level before subjected to functional assays. For siRNA transfection, cells were transfected with the indicated siRNAs using TransIT-X2 (Mirus) for 3^days before being collected for immunoblotting. Cell viability assay Cells were seeded into 96-well plates and / or treated with indicated inhibitors, followed by IncuCyte zoom live-cell analysis (Essen Bioscience) or MTT assay to evaluate cell viability. For IncuCyte analysis, four fields / well were captured, and mean of cell confluence was used to analyze cell viability. For MTT assay, the culture medium was replaced by serum-free medium containing 5 mg / mL 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; M5655, Sigma Aldrich) and was incubated at 37°C. After 2 hours, MTT solution was replaced by dimethyl sulfoxide (DMSO). Plate was gently shaken on an orbital shaker for 30 min at room temperature (RT). The colored solution was quantified by measuring CMUH-0002PCTUS OD570. Data were analyzed using GraphPad Prism 10 and / or visualized by Morpheus software. Clonogenic assay About 1 × 103to 1 × 104cells were seeded into 6-well plates and treated with indicated inhibitors for 1 to 2 weeks. The cell colonies formed were then stained by 1% crystal violet solution prepared in 30% ethanol for 30 minutes and then washed with water to remove excess stain. Images were acquired, and colony numbers were quantified using ImageJ software. Soft agar assay Soft agar assay was performed in 6-well plates. The wells were coated with a base layer of 0.6% low- melting-point agarose in complete medium and covered with a top layer of 0.3% low-melting-point agarose in complete medium containing 1 × 104cells / well. After 4 weeks, the cell colonies were stained with crystal violet (0.01%). The number of colonies was counted by ImageJ software. Transwell migration assay About 2 × 105cells in 200 μL serum-free medium were plated in the upper chamber of Transwell inserts (8 μm pore size, Corning), and 750 μL of 10% FBS-containing medium was put in the lower chamber as a chemo-attractant. After 24 hours, cells were fixed in 4% formaldehyde and stained with crystal violet. Non- migrated cells on the upper chamber were scraped by cotton swabs. The lower chamber containing the migrated cells was captured by Echo Revolve Microscope and then quantified by ImageJ software. Plasmid construction and site-directed mutagenesis The HER2 mutations were introduced into pcDNA6a-HER2-HA (for 293T cells transient transfection) or into pMSCV-HER2-Flag (for retrovirus expression) and MMTV-neuNT (for HER2 transgenic mice) plasmids by the QuikChange II Site-Directed Mutagenesis Kit (Agilent) with specific primers listed in Table 2 according to the manufacturer’s instructions. CMUH-0002PCTUS Table 2. Sequences of the oligonucleotides used in plasmid construction and site-directed mutagenesis CMUH-0002PCTUS F: forward primer; R: reverse primer; Underlined letters indicate mutation sites. 3D spheroid formation The 8-well chamber slide with removable chamber was used for 3D spheroid formation. The wells were coated with 25 μL of Growth Factor Reduced Matrigel (Corning) before adding 400 μL of 2,000 cells in spheroid medium containing 2% Matrigel. Spheroid medium was refreshed every 3 days, and spheroids were captured by Echo Revolve Microscope after 2 weeks for SkBr3 and Sk / LR10 and 4 weeks for BT474 and BT / LR3. The number and size of spheroids were analyzed by ImageJ software. The viable spheroids were determined by CellTiter-Glo 3D Reagent (Promega), following the manufacturer’s instructions, and detected by GloMax Discover Microplate Reader (Promega). Spheroid medium contains serum-free medium supplied with 1% penicillin-streptomycin (P / S), epidermal growth factor (EGF; 20 ng / mL), basic fibroblast growth factor (bFGF; 20 ng / mL), insulin (4 µg / mL), and B27 supplements (Gibco). Whole-mount immunofluorescence staining Organoids / spheroids were fixed by 2% paraformaldehyde and 0.1% glutaraldehyde prepared in phosphate-buffered saline (PBS) for 30 minutes, followed by incubation with the blocking buffer (1% bovine CMUH-0002PCTUS serum albumin (BSA) and 0.2% Triton-X100 in PBS) for 1 hour. Samples then were incubated with primary antibodies overnight at 4oC and secondary antibody for 1 hour at 4°C. After nuclear staining by 4’,6-diamidino- 2-phenylindole (DAPI), slide was covered by mounting medium. Whole-mount spheroid / organoid sections were captured by Andor Dragonfly High Speed Confocal Microscope. The primary antibodies including HER2 (sc-393712, Santa Cruz), HER2 pS1054, SOX2 (3579, Cell Signaling), and Ki67 (MA5-14520, Invitrogen) were used. RNA isolation, RNA-sequencing (RNA-seq), and gene set enrichment analysis (GSEA) Total RNA was isolated from MDA-MB-231 HER2 stable clones stably expressing HER2 WT (n = 3 clones), S1051A (n = 2), S1054A (n = 2), or MSCV vector control (n = 2) using Trizol reagent (15596026, Invitrogen). RNA libraries were prepared and sequenced by GENEWIZ using the Illumina HiSeq platform (2× 150 bp paired end, ≥ 6 Gb per sample). Gene expression was quantified as fragments per kilobase of transcript per million mapped reads (FPKM), and values were normalized to vector controls. These normalized expression values were used for GSEA and Ingenuity Pathway Analysis (IPA) (Qiagen). For GSEA, expression profiles from S1051A and S1054A clones were averaged to represent the HER2 mutant condition. Acetaldehyde dehydrogenase (ALDH) activity assay The ALDH enzyme activity was performed by ALDEFLUOR Kit (Stem Cell Technologies), following the manufacturer’s instructions. Briefly, cells were resuspended in 1 mL assay buffer containing 5 μL of the activated ALDEFLUOR reagent (test tube). Immediately, 500 μL of mixture were transferred into new tube containing 5 μL of the ALDEFLUOR DEAB reagent (stop solution; control tube). Both tubes were incubated for 60 minutes at 30°C and then centrifuged to remove staining solution. The pellet was resuspended in assay buffer and then subjected to flow cytometry. HER2 in vitro kinase assay Human recombinant HER2 (676-end) was obtained from Promega (V9381) and utilized as the kinase source in the in vitro kinase assay. The assay utilized a kinase buffer containing 40 mM Tris (pH 7.5), 20 mM MgCl2, 0.1 mg / mL BSA, and 50 µM DTT. The reaction mixture comprising 50 µM ATP / GTP, 100 ng HER2 CMUH-0002PCTUS kinase, and 1 µg substrate (or 0.5 to 2 µg as indicated) was incubated at room temperature (25°C) for 90 minutes. After the kinase reaction incubation, 1 μL of reaction was spotted on nitrocellulose (NC) membrane for dot blot assay to detect with indicated antibodies and subjected to adenosine diphosphate (ADP) detection with ADP-Glo Kinase Assay (V6930, Promega) for bioluminescent detection of a kinase activity, following the manufacturer’s instructions. The luminescent signal was detected as relative light units (RLU) by GloMax Discover Microplate Reader (Promega). HER2 peptides were used as substrate, as listed below in Table 3. Polypeptide containing glutamine and tyrosine residues in a 4:1 ratio was used as a positive control for kinase reaction. Table 3. HER2 peptides and their amino acid sequences used as substrate for in vitro kinase assay Underlined letters indicate mutation sites. Surface plasmon resonance (SPR) SPR experiments were conducted using a Biacore T200 instrument (Cytiva) at 25oC. Recombinant HER2 protein (25 ng / μL) was immobilized onto a CM5 sensor chip via amine coupling using 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and ethanolamine in PBS running buffer. A final immobilization level of 15,000 resonance units (RU) was achieved. Binding assays were CMUH-0002PCTUS performed using AMPPNP and GMPPCP as analytes prepared in 1.05× PBS-P (PBS with 0.05% Tween-20) supplemented with 1 mM MgCl2. Analytes were injected at 30 μL / min flow rate for 120 s association and 120 s dissociation phases. Surface was regenerated between cycles with glycine-HCl (pH 2.0) for 30 s at a flow rate of 30 μL / min. Steady-state binding analyses were performed using Biacore Evaluation Software (Cytiva). Equilibrium dissociation constants (KD) were determined by fitting the levels of binding response against a series of analyte concentrations using a steady-state affinity model. The model fitting with a Chi squared (χ²) value below 2% of Rmax is considered acceptable. Fluorescence-based thermal shift assay (TSA) Fluorescence-based thermal shift assay was performed as described in Zhuang, C. et al. (J. Med. Chem. (2014) 57, 1121-1126). Briefly, 5 mg of HER2 kinase was incubated with or without 1 mM HER2 S peptide in the presence of 1× SYPRO orange dye (S6650, Invitrogen) for 20 minutes at 4oC. The reaction was prepared in MicroAmp Optical 96-well reaction plate and then applied to Applied Biosystems Quant Studio 7 PCR machine to detect the fluorescence intensity at an excitation of 465 nm and emission of 580 nm when the temperature increased 1°C / min from 25°C to 95°C. Bio-layer interferometry (BLI) assay The BLI assay was performed to determine the binding affinity of guanosine triphosphate (GTP) in HER2 by the wavelength shift of the white light on the Octet RED96 system (ForteBio). The HER2 (676- 1255)-glutathione S-transferase (GST) fusion protein was immobilized on the surface of biosensor chip coated with anti-GST (18-5096, ForteBio) in an overnight incubation at 4oC. The serial dilutions of GTP were prepared in 200 μL assay buffer. Prior to starting the assay, the samples and the instrument were warmed up to room temperature (RT). The wavelength shift (nm / s) was detected for 120 seconds as binding rate. Animal study The embryonic stem cell-based gene targeting method was used to generate HER2 transgenic mice bearing HER2 wild type (WT) and HER22A mutant. pMMTV-neuNT plasmid was purchased from Addgene (#1823), and HER22A mutant was generated using the QuikChange II Site-Directed Mutagenesis Kit (Agilent). DNA for microinjection was prepared as described in Muller, W. J. et al. (Cell (1988) 54, 105-115), and mice CMUH-0002PCTUS were generated by the Transgenic Mouse Core Lab of National Taiwan University Centers of Genomic and Precision Medicine. For lapatinib-relapse HER2-transgenic mouse model, tumor was first removed by surgery when tumor volume reached 100 mm3. Mice were then treated with lapatinib at 10 mg / kg daily by oral gavage until tumor recurrence. Recurrent tumors were harvested for organoid culture or formalin-fixed, paraffin-embedded for immunohistochemical (IHC) analysis. Tumor volume was measured following the formula: large diameter × (small diameter)2 / 2. For drug testing, HER2 WT transgenic mice underwent tumor resection and were then randomized to receive lapatinib (10 mg / kg) or compound 142539 (10 mg / kg) by daily oral gavage. Tumor volume was measured at regular intervals. Mouse model of brain metastasis from intracarotid inoculation was performed as described by Zhang, C. et al. (J. Vis. Exp.8:55085). About 5 × 105cells of MDA-MB-231 overexpressing HER2-WT / 2A were injected into the carotid artery of female non-obese diabetic / severe combined immunodeficiency (NOD / SCID) mice (8-week-old). The bioluminescence signal was monitored with an in vivo optical imaging system. Spheroid formation and organoid culture For spheroid formation, 1,000 cells in 500 μL of medium were embedded in 50% growth factor reduced Matrigel (Corning) prepared in stem cell medium (1% P / S, 20 ng / mL EGF, 20 ng / mL bFGF, 4 µg / mL insulin, and 1× B27). The mixture was seeded into 24-well ultra-low attachment plates. Spheroid medium was refreshed every 3^days. Spheroids were imaged after 2 weeks for SkBr3, Sk / LR10, and MDA-MB-231 cells overexpressing HER2, and after 4^weeks for BT474 and BT / LR3 cells. Spheroid number and size were quantified using ImageJ software. Spheroid viability was assessed using CellTiter-Glo 3D reagent (Promega) according to the manufacturer’s instructions. For plate-based spheroid formation, 500 cells were seeded into 96-well U-bottom ultra-low attachment plates and incubated for 7^days. Mouse tumor organoid culture was performed as described by Sachs, N. et al. (Cell (2018) 172, 373-386 e310). Briefly, tumors were minced in AdDF+++ medium before digested in BC organoid medium containing 2 mg / mL collagenase (C9407, Sigma) for 2 hours at 37°C. The digested tissue was strained through 70 mM CMUH-0002PCTUS strainer, followed by centrifugation at 400 ×g for 5 minutes. The pellet was then incubated in red blood cell lysis buffer (11814389001, Roche) for 5 minutes at room temperature (RT) to lyse erythrocytes and washed by AdDF+++ medium. The pellet was resuspended in 40 mL of basement membrane extract (3533-010-02, Trevigen) and plated on 24-well plate ultra-low attachment surface (3473, Corning). After gelation, 400 mL of BC organoid were added and refreshed every 4 days. For drug screening, 5 μL of organoid (75 organoids per μL) were seeded into 96-well ultra-low attachment plates as described in S. Loibl et al., (Lancet (2017) 389, 2415-2429), and compounds were added accordingly. Organoid viability was detected using CellTiter-Glo 3D reagent (Promega). AdDF+++ medium contains Advanced DMEM / F12 (12634-034, Invitrogen), 10 mM HEPES, 1× GlutaMAX supplement (12634-034, Invitrogen), and 50 mg / mL primocin (Ant-pm-1, Invitrogen). BC organoid contains 50% AdDF+++ medium, 50% L-WRN conditional medium, 5 nM Neuregulin 1 (100-03, Peprotech), 5 ng / mL FGF 7 (100-19, Peprotech), 20 ng / mL FGF10 (100-26, Peprotech), 5 ng / mL EGF (AF- 100-15, Peprotech), 500 nM A83-01 (2929, Tocris), 5 mM Y-27632 (Y-27632, Abmole), 500 nM SB202190 (S7067, Sigma), 1× B27 supplement (17504-44, Gibco), 1.25 mM N-acetylcysteine (A9165, Sigma), 5 mM nicotinamide (N0636, Sigma), 100 U / mL penicillin, and 100 mg / mL streptomycin (15140-122, Invitrogen). The conditioned medium was produced from L-WRN cell lines as described by Miyoshi, H. & Stappenbeck, T. S. (Nat. Protoc. (2013) 8, 2471-2482). Dissemination in brain extracellular matrix Decellularization of porcine brain tissue and preparation of brain extracellular matrix (B-ECM) hydrogel were performed as previously described by Z. L. Cai et al. (Am. J. Cancer Res. (2023) 13, 3266-3274). Cells (1 × 106) were suspended in 100 μL of solubilized B-ECM, and 0.5 μL of the cell-B-ECM mixture were seeded as domes into 96-well plates. Domes were imaged daily to monitor invasion fronts over time. Cell growth and proliferation within the B-ECM hydrogel were quantified using the CellTiter-Glo 3D assay (Promega) according to the manufacturer’s instructions. Clinical samples and immunohistochemical (IHC) staining, Opal multiplex IHC, and image cytometry Patient-matched primary and recurrent tumor samples from HER2+ BC patients who received Herceptin CMUH-0002PCTUS as the first-line treatment were obtained from China Medical University Hospital with informed consent (IRB approval CMUH107-REC1-062). BC tissue array was obtained from China Medical University, and GC tissue array was obtained from Kaohsiung University. A total of 106 HER2+ BC patients were enrolled from China Medical University (CMU Hospital (CMU, n = 78)) and E-DA Hospital (EDA, n = 28) in Taiwan (CMU / EDA cohort), including 34 matched pairs of tumors and adjacent normal tissues. An additional 177 HER2+ GC patients were collected from Kaohsiung Medical University Hospital (KMU cohort, Taiwan), with 152 matched tumor-normal tissue pairs. Paraffin-embedded sections were deparaffinized by xylene rehydrated with gradient of alcohol. Immunohistochemical staining utilized the UltraVision Quanto Detection System HRP DAB kit (TL-125-QHD, Epredia) following the manufacturer’s instructions. The primary antibodies used for IHC staining include: HER2 (ab214275, Abcam), HER2 pY1221 / 2 (2243, Cell Signaling), HER2 pS1054, Ki67 (MA5-14520, Thermo Fisher), E-cadherin (3195, Cell Signaling), vimentin (5741, Cell Signaling), and SOX2 (3728, Cell Signaling). For Opal multiplex IHC (NEL810001KT, Akoya Biosciences), the manufacturer’s instructions were followed. The primary antibody-Opal fluorophore pair was applied in the sequence of pS1054 / Opal 520, HER2 (ab214275, Abcam) / Opal 570, CK19 (ab52625, Abcam) / Opal 690, and DAPI. The slides were scanned by TissueFAXS microscope system (TissueGnostics), and the levels of HER2 pS1054, total HER2, and CK19 were quantified by StrataQuest software. Additionally, 292 primary tumor tissues (pre- and post-Herceptin treatment) and 56 metastatic tumor samples were obtained from 146 HER2+ BC patients treated at Xiangya Hospital (Xiangya cohort, China) to assess HER2 pS1054 expression. Immunohistochemical staining was performed as described above. Briefly, tissue sections were incubated with anti-pS1054 antibody, followed by biotin-conjugated secondary antibody (P0615, Beyotime Biotechnology) and avidin-biotin-peroxidase complex. Herceptin treatment response was classified based on RECIST (Response Evaluation Criteria in Solid Tumors): complete and partial responses were defined as Herceptin-sensitive, whereas stable and progressive disease were classified as Herceptin- resistant. Overall survival analysis was performed on 47 patients who received Herceptin treatment, stratified CMUH-0002PCTUS by pS1054 expression levels. The intensity of IHC staining was evaluated as: negative (-), weak (+), moderate (++), strong (+++ or ++++). Structure-based virtual screening Structure-based virtual screening was conducted using the docking software LeadIT. The crystal structure of HER2 kinase domain (PDB ID: 3RCD) was obtained from the Protein Data Bank. The protein structure was prepared using LeadIT, and all water molecules were removed. Approximately 280,000 compounds from the NCI library were selected for the screening. Docking was performed in LeadIT using default settings. High-content imaging HER2+ BC cells (SkBr3; 8,000 cells per well) were seeded into 96-well half-area, high-content imaging plates with film bottoms (Corning, 4680). After 24^h, cells were treated with 56 candidate compounds at 10 μM for 24 h. Cells were then fixed with 4% paraformaldehyde and blocked in PBS containing 1% BSA. Samples were incubated overnight at 4oC with primary antibodies against HER2 and pS1054, followed by fluorophore-conjugated secondary antibodies for 1 h at room temperature. Nuclei were counterstained with DAPI. Plates were imaged using a high-content imaging system, and fluorescence intensities of HER2 and pS1054 were quantified. Data analysis All statistical analyses were performed using GraphPad Prism v8.0. Heatmaps were generated using Morpheus software. Statistical tests used included paired or unpaired two-tailed Student’s t-test, two-way ANOVA, log-rank (Mantel-Cox) test for Kaplan-Meier survival analysis, and Pearson’s χ² test for association between Herceptin response and pS1054 status. Data are presented as mean ± standard deviation (SD). Statistical significance was defined as p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****). Example 1: Absence of tyrosine phosphorylation in resistant HER2+ tumors In an HER2-transgenic mouse model with 140-days treatment of lapatinib, the higher proliferative (Ki67- CMUH-0002PCTUS positive) recurrent tumors still presented high expression levels of HER2 protein, but the phosphorylation of HER2 at Tyr1221 / 1222 residues was not observed as shown in FIG. 1A. Similar observations of non- phosphorylation of HER2 at Tyr1221 / 1222 residues were also found in cancer cells, such as acquired lapatinib- resistant (LR) clones of HER2+ BT474 clone (BT / LR3) and acquired LR clones of SkBr3 clone (Sk / LR10), as shown in FIG. 1B. However, when HER2 expression was silenced by shRNAs, e.g., shHER2#A and shHER2#B (FIG.1C), viability (FIG. 1D), colony formation (FIG. 1E), 3D spheroid formation (FIG. 1F), and migration (FIG. 1G) of the BT / LR3 and Sk / LR10 clones were all found to be reduced, similar to their non-lapatinib-resistant parental cells, suggesting that HER2 protein remains playing a role for the oncogenic functions. The protein tyrosine phosphorylations of most receptor tyrosine kinases (RTK) (FIG. 1H) and anti-HER2 immunoprecipitates (FIG.1I) were not detected in the resistant LR clones. These results indicate that HER2 in the resistant cells does not present tyrosine phosphorylation but still possesses oncogenic functions to promote cancer progression. Example 2: HER2 S1051 (pS1051) and S1054 (pS1054) are associated with drug resistance to HER2- targeted therapies Using an anti-phospho-Akt substrate (PAS) antibody, a signal of potential HER2 serine phosphorylations within the RXRXXS / T consensus sequence was detected and found to be increased in the anti-HER2 immunoprecipitates from resistant HER2+ cancer cell clones BT / LR3 and Sk / LR10, as shown in FIG.2A, and also in lapatinib-treated parental BT474 and SkBr3 clones as shown in FIG. 2B. These HER2 serine phosphorylations were found to be removable by in vitro treatments with phosphatase, suggesting that some unidentified HER2 serine phosphorylations could be induced by lapatinib. In mass spectrum analysis, the phosphorylations of HER2 at S1051 (pS1051) and S1054 (pS1054) were identified in BT / LR3 cells (FIG.2C). S1051 matches the RXRXXS / T consensus sequence, and the neighboring serine cluster is conserved in HER2 among diverse species (FIG. 2D); however, this homologous sequence was not found in other members of the epidermal growth factor receptor (EGFR) family (FIG.2E). To further investigate these two pS1051 and pS1054 sites as specific phosphorylations associated with CMUH-0002PCTUS drug resistance, phospho-specific antibodies against HER2 pS1051 and pS1054 were generated and validated by peptide competition (FIG.2F). They were also confirmed with phospho-ablative alanine mutations S1051A, S1054A, or both S1051A and S1054A (2A) (FIG.2G). It was found that mutation of HER2 S1051 to alanine also abolished HER2 pS1054, suggesting a subsequent phosphorylation event at S1054 following S1051. However, only the pS1054 antibody works for IHC (FIG.2H) and IF (FIG.2I). Both antibodies showed no cross-reactivity with other EGFR members (FIG. 2J). It is noted that the HER2 mutations of the novel S1051A and S1054A phosphorylation sites to Ala (2A) did not alter tyrosine phosphorylation at Y1221 / 2, and conversely, HER29F mutation (phenylalanine substitution at nine autophosphorylation sites, Tyr877, Tyr1005, Tyr1023, Tyr1112, Tyr1139, Tyr1196, Tyr1221, Tyr1222, and Tyr1248) did not affect pS1051 / 1054 levels (FIG. 2K), indicating that these phosphorylations were independent. Furthermore, the 2A mutation did not impair Erk activation, whereas the 9F mutation did, suggesting that HER2 serine phosphorylation may signal through distinct downstream pathways from its canonical tyrosine phosphorylation. Despite suppression of pY1221 / 2, short-term treatment with and acquired resistance to HER2 tyrosine kinase inhibitors (TKIs) lapatinib (Lap) and tucatinib (Tu) increased HER2 pS1051 / 1054 in HER2+ breast cancer cells, including BT474, SkBr3 and SUM190 cells, along with their lapatinib-resistant (LR) and tucatinib-resistant (TuR) derivatives (FIG. 2L). These phosphorylations were also elevated in Herceptin- resistant cells (BT / HR) and were further enhanced by lapatinib (FIG.2M). These findings suggested that HER2 pS1051 / 1054 could be a common mechanism contributing to resistance against HER2-targeted therapies. Namely, the correlation of HER2 pS1051 / 1054 with drug resistance to both HER2 antibodies and TKIs indicates that these serine phosphorylations contribute to the development of drug resistance to HER2-targeted therapies. Example 3: HER2 pS1054 correlates with poorer prognosis of HER2+ BC and GC and links to HER2+ BC progression HER2 pS1054 levels were higher in tumor tissues of HER2+ BC (FIG.3A) and GC (FIG.3B) compared to adjacent normal tissues with a positive correlation with the cancerous marker cytokeratin 19 (CK19) in HER2+ BC tumors (FIG. 3C). IHC staining of pS1054 in tumor tissues shows that HER2 pS1054 exists in a CMUH-0002PCTUS partial population within HER2-overexpressing tumors (FIG. 3D) and is not completely in accordance with HER2 protein expression (FIG. 3E). Additionally, cases with high HER2 pS1054 were identified as HER2 pS1054-positive, defined as IHC staining intensity of 2+ or higher (FIG.3F), and associated with a poorer overall survival rate in breast cancer (FIG.3G) and in gastric cancer (FIG. 3H). HER2 pS1054 status also reflected the differential response to Herceptin treatment. Among pre-treatment patients, HER2 pS1054-positive cases are 87.3% in Herceptin-resistant and 52.8% in HER2-sensitive patients (FIG. 3I), and higher HER2 pS1054 was significantly associated with poor response to Herceptin (FIG. 3J). Furthermore, HER2 pS1054 positivity was linked to reduced overall survival in patients receiving Herceptin (FIG. 3K). Furthermore, it was also found that Herceptin treatment suppressed HER2 pTyr1221 / 1222 but not pS1054 in human HER2-positive breast tumors (FIG.3L). Moreover, HER2 pS1054 high expression was also detected in all examined metastatic lesions, including brain metastasis, lung metastasis, liver metastasis, lymph node metastasis, and chest wall metastasis (FIGs.3M and 3N). Among brain metastases, 18 of 26 cases showed strong HER2 pS1054 positivity (+++ or ++++), suggesting its enrichment in brain metastatic sites. Therefore, HER2 pS1504 may confer Herceptin resistance and fuel brain metastasis. HER2 pS1054 was further evaluated in Herceptin-recurrent brain metastatic tumors (FIG.3O) as well as lung and liver metastatic tumors (FIG. 3P), and all of the above found elevated levels of HER2 pS1054, indicating that pS1054 may be associated with resistance to HER2-targeted therapies and tumor metastasis. Example 4: HER2 pS1051 / 1054 drives cancer stemness and mutations at HER2 S1051 / 1054 suppress tumor formation Current HER2-targeted therapies show limited efficacy against cancer stem cells (CSCs), which drive resistance and metastasis. CSCs, enriched in HER2+ BC, exhibit self-renewal capacity, and markers such as SOX2, ALDH, and CD44+ / CD24- are associated with increased migration and metastatic potential. Aberrant HER2 signaling promotes CSC traits via epithelial-mesenchymal transition (EMT), Notch, Wnt / β-catenin, and PI3K / AKT pathways, contributing to tumor initiation, therapy resistance, and brain metastasis. It is noted that CMUH-0002PCTUS TKIs and ADCs fail to eliminate CSCs, as shown by persistent mammary spheroid formation in vitro in recent reports. These findings suggest that HER2’s role in sustaining CSC traits extends beyond its canonical tyrosine kinase activity. The impact of pS1051 / 1054 on CSC, a key driver of therapeutic resistance and tumor dissemination, was examined. The LR clones with elevated HER2 pS1051 / 1054 showed increased spheroid-forming capacity (FIGs. 4A and 4B) and elevated expression of stem cell marker SOX2 (FIG. 4C) compared to parental cells. This effect was diminished upon withdrawal of lapatinib treatment (FIGs.4D and 4E). Moreover, the reduction of SOX2 and HER2 pS1051 / 1054, along with the recovery of HER2 pTyr and its downstream Akt / ERK pathways, was also found in different LR clones when lapatinib treatment was withdrawn (FIGs.4E and 4F), suggesting that the HER2-induced spheroid formation relies on pS1051 / 1054. These findings indicated that HER2 pS1051 / 1054 but not pY1221 / 2 correlated with stem-like features, implying that lapatinib-induced HER2 pS1051 / 1054 might contribute to stem-like features. In line of this concept, gene set enrichment analysis (GSEA) revealed that HER2-induced expression of gene sets associated with stem cell proliferation (FIG. 4G), mammary gland development (FIG. 4H), and Mueller plurinet (FIG.4I) were all reduced when both HER2 S1051 / 1054 were mutated to Ala, suggesting the promoting effects of HER2 pS1051 / 1054 on cancer stemness. In addition, single mutation at either S1051 or S1054 also repressed HER2-induced gene expressions involved in stem cell proliferation such as ID2, ID4, ALDH1A3, NOG, FZD8, TGFB1, TGFA, and FOXO1 (labeled with “*” in FIG.4J). Consistent with transcriptomic changes, mutations of HER2 S1051 / 1054 (S1051A, S1054A, and 2A) generated to mimic non-phosphorylation of these serine sites found that HER2-induced expressions of SOX2, ALDH1, and CD44 cancer stem cell markers (FIG. 4K), ALDH activity (FIG. 4L), anchorage-independent growth (FIG.4M), and 3D spheroid formation (FIG.4N) were all nearly abolished. To evaluate the functional role of HER2 S1051 / 1054 phosphorylation in tumor initiation, MMTV-driven HER2-transgenic mice harboring the 2A mutant were generated. These mice developed tumors with delayed onset (FIG.4O) and reduced tumor burden compared to HER2-WT control mice (FIG.4P). Organoids derived from 2A tumors of transgenic mice showed impaired growth (FIG.4Q), associated with upregulation of tumor CMUH-0002PCTUS suppressor E-cadherin and downregulations of CSC marker SOX2, metastatic marker Vimentin, and proliferative marker Ki67 (FIG. 4R). Together, these findings demonstrate that HER2 S1051 and S1054 phosphorylation supports cancer stemness and tumor initiation. Example 5: HER2 pS1051 / 1054 promotes brain metastasis Consistent with enhanced spheroid formation, LR clones also showed increased growth (FIGs. 5A and 5B) and migration (FIG. 5C) in the 3D culture condition with extracellular matrix (ECM) extracted from the pig brain. HER22A mutation was found to also suppress expressions of gene sets involved in cell migration, cell invasion, angiogenesis, and neurological disorders, and those related to cell morphogenesis involved in neuron diseases and differentiation (FIGs. 5D and 5E). In addition, IPA also revealed that HER2 2A mutant downregulated pathways associated with fatty acid synthesis and polyunsaturated fatty acid biosynthesis (FIG. 5D). To assess whether HER2 pS1051 / 1054 contributes to brain colonization, MDA-MB-231 cells stably expressing HER2 WT or 2A mutant were embedded in porcine B-ECM to evaluate invasion and expansion of cancer cells. As shown in FIGs.5F to 5H, the dissemination of tumor cells from the dome (white dashed circle) was monitored over 5 days, and cell spreading was quantified (red dashed line). Cells expressing HER22A displayed significantly reduced dissemination area and lower viability compared to HER2 WT controls, suggesting that HER2 pS1051 / 1054 promotes tumor cell adaptation and outgrowth in B-ECM. Additionally, the 2A mutation at HER2 S1051 / 1054 also suppressed brain metastasis of HER2-overexpressing breast cancer cells in NOD-SCID mice through internal carotid artery injection (FIG.5I). These results indicate that HER2 pS1051 / 1054 mediates tumor initiation and brain metastasis. Example 6: HER2 possesses a serine kinase activity for HER2 S1051 / 1054 autophosphorylations using ATP / GTP as phosphor-donors HER2 S1051 / 1054 matches the conserved substrate motif for several Ser / Thr kinases, such as AKT and SGK (FIG.6A). However, their phosphorylations were not affected by the pharmacological inhibitors or gene silencing of more than ten tested Ser / Thr kinases, including Akt / protein kinase B signaling inhibitor-2 (API- CMUH-0002PCTUS 2), an inhibitor of Akt (FIG. 6B), the inducer of transforming growth factor beta (TGF-β) type II receptor degradation-1 (ITD-1; FIG.6C), shRNAs (shPKA#1, shPKA#2, and shPKA#3, as listed in Table 4 below) that silence protein kinase A (PKA) (FIG. 6D), and H89, an inhibitor of PKA (FIG.6E). Table 4. shRNA sequences used for silencing protein kinase A Potential kinases, predicted to mediate pS1051 (such as PHKG1, PKCα, PKCβ, and PKCδ) and pS1054 (such as GAK, PBK, BIKE, and AAK1) in PhosphositePlus, mostly were suppressed in LR cells except for PKCδ and PBK (FIG.6F). However, sotrataurin, a pan-PKC inhibitor, did not reduce pS1051 / 1054 (FIG.6G). Silencing PKCδ and PBK with siRNAs listed in Table 5 below also did not suppress these phosphorylations (FIGs. 6H and 6I). These findings suggest that these phosphorylations may be induced by non-canonical serine / threonine kinases. Table 5. siRNA sequences used for silencing PKCδ and PBK *: SASI_Hs01_00061170 (MilliporeSigma); **: SASI_Hs01_00225165 (MilliporeSigma); F: forward; R: reverse. The kinase-dead (K753M), ATP-binding deficient (KGG; K753M, G729D, G732D), and magnesium- CMUH-0002PCTUS binding deficient (D863K) mutants of HER2 dramatically reduced HER2 pS1051 / 1054 as well as pY1221 / 1222 (FIG. 7A). In vitro kinase (IVK) assays were performed using purified HER2 kinase domain with ATP as phospho-donor, and synthetic peptides were used as substrates: the 2S peptide containing HER2 S1051 / 1054, the 2Y peptide containing Y1221 / 2, and the negative control (NC) peptide containing a scrambled sequence of the 2S peptide. HER2 can auto-phosphorylate not only tyrosine residues (FIG.7B, bar 3), but also serine residues (FIG.7B, bar 5). Further, it was also unexpectedly observed that lapatinib did not suppress (nor induce) ATP-dependent pS1051 / 1054 in IVK assays (FIG.7B, red bars), whereas ex vivo cell models showed an increase in pS1051 / 1054 by lapatinib as shown in the above examples. These results suggested that additional cellular components or mechanisms may contribute to lapatinib-induced pS1051 / 1054 in cell models. GTP can serve as a co-substrate for serine kinases. In IVK assays, HER2 primarily utilized ATP to catalyze pTyr1221 / 2, but supported pS1054 by using both ATP and GTP (FIG. 7C, lane 2 and lane 7). While adenylyl-imidodiphosphate (AMPPNP), the non-hydrolyzable ATP (nATP) analog, and lapatinib effectively suppressed ATP-mediated pY1221 / 2 (FIG.7C, lane 9 and lane 10), they unexpectedly enhanced GTP-mediated pS1054 (FIG. 7C, lane 3 and lane 5). In contrast, guanosine-5’-[β,γ-methylene]triphosphate (GMPPCP), the non-hydrolyzable GTP (nGTP), competitively inhibited GTP-driven-pS1054 but not ATP-driven-pS1054 (FIG. 7C, lane 4 and lane 8). Surface plasmon resonance (SPR) analyses revealed that nGTP bound to HER2 with KD ≈ 90 nM (FIG. 7D), comparable to nATP (KD ≈ 58 nM, FIG.7E). Similar to ATP which requires magnesium as metal cofactor, GTP binding required Mg²⁺, as the absence of Mg²⁺ reduced binding affinity of nGTP bound to HER21000 times (KD ≈ 96 µM) (FIG.7F). Additionally, both purified HER2 kinase domain (HER2676-1045) (FIG.7G) and HA-tagged full length HER2 from HEK-293T overexpressing cells (FIG. 7H) bound more strongly to GTP-conjugated than ATP- conjugated agarose beads even higher volumes of ATP-agarose and total lysate were used. Interestingly, while lapatinib expectedly decreased the interaction between ATP and HA-tagged HER2, it paradoxically enhanced GTP-HER2 interaction, suggesting a lapatinib-induced shift in HER2 nucleotide binding preference (FIG.7I). In agreement with the above result, the purified HER2 kinase domain had a strong binding affinity with CMUH-0002PCTUS HER2 S1051 / 1054-containing peptide in the protein thermal shift assay (FIG.7J). Furthermore, in vitro kinase assays revealed that the purified HER2 kinase domain phosphorylated HER2 peptides containing S1051 / 1054 residues using ATP as a phosphor-donor, and these phosphorylations were abolished by the corresponding peptides with the 2A mutations at HER2 S1051 / 1054 (FIG.7K). Molecular docking of GTP / Mg2+and lapatinib to HER2 by Proteinix Server (AlphaFold 3-based) showed that lapatinib bound to ATP binding site and GTP was found at the outskirt of ATP binding site with Mg2+involved in coordination of GTP binding, particularly the triphosphate group (FIG. 7L, right). The residues within 4 Å away from GTP are presented as sticks (FIG. 7L, left). Among these residues, the ATP‑binding deficient KGG mutant and the Mg²⁺‑binding deficient D863K mutant, but not the kinase‑dead K753M mutant, abolished lapatinib‑induced pS1051 / 1054 (FIG. 7M). Moreover, P885, the first residue of the activation segment (PIKWMALE), involves in positioning tyrosyl substrates, whereas W888, which interacts with E914 to stabilize the open conformation, may be involved in seryl substrate positioning (FIG.7N). These findings suggest that beyond working as a tyrosine kinase, HER2 may also function as a serine kinase for autophosphorylation at S1051 / 1054 with dual usage of ATP or GTP as phosphate donors. These findings support the notion that HER2 may utilize both ATP and GTP as phosphate donors for S1054 phosphorylation through distinct mechanisms. This dual specificity may underlie an adaptive mechanism in which enhanced GTP responsiveness serves as a trade-off to compensate for inhibition by ATP-competitive inhibitors. Example 7: HER2 symmetric dimer drives serine autophosphorylation HER2 activation relies on homo- or hetero-dimerization with other EGFR-family receptors, adopting distinct orientations. The I714Q mutation, which disrupts asymmetric dimerization, moderately reduced basal HER2 pS1051 / 1054 (FIG. 8A, left). In contrast, the IRVR (I748R / V750R) and NRKF (N764R / K765F) mutations, which impair symmetric EGFR-like and HER3-like dimerization, respectively, significantly reduced basal pS1051 / 1054 levels (FIG. 8A, middle and right). Interestingly, upon lapatinib treatment, pS1051 / 1054 levels were further increased in WT, I714Q, and NRKF mutants, but not in IRVR (FIG. 8B). Similar inhibitory effects of IRVR mutation on lapatinib-induced HER2 dimerization were found by using CMUH-0002PCTUS native PAGE (FIG. 8C). The results indicated that symmetric-EGFR like dimerization was required for lapatinib-induced HER2 pS1051 / 1054. Once IRVR mutation impaired symmetric-EGFR like dimerization, HER2 pS1051 / 1054 was no longer responsive to the lapatinib induction. Lapatinib has previously been shown to induce symmetric HER2-HER3 heterodimers. However, in LR clones, EGFR, HER3, and HER4 were not activated in LR clones, and silencing of EGFR, HER3, or HER4 did not alter HER2 pS1051 / 1054 levels (FIG. 8D). These results strongly support that a symmetric HER2- HER2 homodimer conformation is required for S1051 and S1054 autophosphorylations. Since phosphorylation requires substrate binding to the kinase domain, the binding activity of 2S and 2Y peptides to the HER2 kinase domain was then examined. IVK competition assays were carried out comparing 2A (S1051 / 1054A) versus (vs.) 2S, 2F (Y1221 / 2F) vs.2S, 2F vs.2Y, and 2A vs.2Y peptides. The 2A peptide dramatically inhibited phosphorylation of the 2S peptide in a dose-dependent manner, whereas 2F had no such effect (FIG. 8E). Conversely, 2F selectively suppressed phosphorylation of the 2Y peptide, while 2A did not (FIG.8F). These findings support that HER2 functions as a dual-specificity kinase (DSK) that phosphorylates both serine and tyrosine residues. Example 8: HER2 dual-specificity kinase inhibitors (DSKI) show a superior anti-cancer activity than HER2 TKIs In this disclosure, it was found that both tyrosine and serine kinase activities are involved in the oncogenic function of HER2. In this example, it is shown that targeting both Tyr / Ser autophosphorylations of HER2 at its substrate-binding site provides a superior anti-cancer activity with less drug resistance. A screening of compounds in the National Cancer Institute (NCI) library is shown with steps in FIG.9A. Potential candidates with HER2 dual-specificity kinase inhibition were identified, and their inhibitory effects on HER2 pS1051 / 1054 phosphorylation and cancer cell viability were assessed using a high-content screening system (FIG. 9B). Among these candidates, PAN-430 and PAN-467 were used as exemplified examples to demonstrate superior inhibitory effects (IC50< 10 μM) on cell viability (FIG.9C) and spheroid formation (FIG.9D) across parental, lapatinib- and / or Herceptin-resistant cells, compared to lapatinib and tucatinib. Further analysis CMUH-0002PCTUS showed that both compounds directly bound to the HER2 kinase domain (FIG. 9E) and inhibited not only HER2 pTyr1221 / 2 but also pS1051 / 1054 (FIG.9F). Furthermore, HER2 phosphorylation at Serine 1054 was suppressed when GTP was used as the phosphor-donor in in vitro kinase assays (FIG. 9G). In this example, PAN-467 reduced lapatinib-elicited HER2 pS1054 (FIG.9H) and showed effective suppression of both kinase activities with more preference for serine kinase (FIG.9I). Moreover, PAN-467 also induced cell death, as indicated by PI-positive staining, in tumor organoids derived from the HER2-Tg mouse after 48 hours of treatment (FIG.9J). PAN-430 and PAN-467 demonstrated a near abolishment of the viability of tumor organoids after 12 days of treatment, a response not achieved by two HER2 TKIs, lapatinib and tucatinib (FIG.9K). Furthermore, treatment with PAN-467 showed a significantly improved tumor-free survival rate (FIG. 9L) and tumor growth suppression (FIG. 9M) compared to lapatinib in HER2-Tg mice after the surgical removal of the primary tumor. These results indicate that targeting HER2 DSK activity is an effective treatment strategy against HER2+ cancers. Example 9: Molecular docking analysis of HER2 kinase domain for tyrosine / serine phosphorylation This example provides molecular docking analysis result on HER2 kinase domain and reveals the interaction of a serine-containing peptide, which matches the Akt consensus substrate sequence (AKT13CQU peptide), with the substrate binding site in the HER2 kinase domain (PDB ID: 3rcd) (FIG.10A). In the analysis, it was found that serine substrate peptides typically contain positive-charge R / K / H residues in front of serine / threonine residues (FIGs. 10B and 10C). Similar to two serine / threonine kinases, Akt and PIM1, the HER2 kinase domain utilizes negative-charge D / E residues to form hydrogen bonds with the conserved R / K / H residues on the serine substrate peptides (FIGs.10A, 10B, and 10C). In contrast, tyrosine substrate peptides typically contain negative-charge D / E residues in front of tyrosine residue. Receptor tyrosine kinases, including HER2, EGFR, and INSR, utilize positive-charge R / K / H residues to form hydrogen bonds with the conserved D / E residues on the tyrosine substrate peptides (FIGs.10D, 10E, and 10F). Several residues of HER2 kinase domain, including E914, D808, and D845, interact with the serine CMUH-0002PCTUS peptide in the molecular docking analysis. The side chain carboxylic acid of D808 and E914 residues interact with the side chain guanidinium group of -3 and -5 Arg of Ser substrate peptides (FIG. 10G). Charge-switch mutations of D808K and E914K almost abolish the HER2 pS1051 and pS1054 (FIG.10H). The D845 residue, located in the HRD motif in the HER2 kinase domain, acts as a conventional catalytic base to specifically receive a proton from the OH group of tyrosine residue for the catalyzation of phosphorylation. Although the OH group of S1051 residue in the substrate peptide also interacts with the side chain carboxylic acid of D845 (FIG. 10G), mutation at this residue only affects pTyr but not pSer (FIG. 10H), suggesting that D845 in the HRD motif is specific for phosphoryl transfer to tyrosine but not serine. Instead of regulating substrate binding activity, D863 coordinates Mg2+necessary for ATP binding activity of HER2 (FIG.7A), and mutation of D863 residue substantially abolishes both pTyr and pSer (FIG. 10H), suggesting that this residue also mediates the GTP / ATP-binding for pSer. The molecular docking analysis results provided by this example suggest that the substrate-recognition sites for these two types of phosphorylations, namely serine phosphorylation and tyrosine phosphorylation, are overlapped but not identical in the HER2 kinase domain. While some of the embodiments of the present disclosure have been described in detail in the above, it is, however, possible for those of ordinary skill in the art to make various modifications and changes to the embodiments shown without substantially departing from the teaching of the present disclosure. Such modifications and changes are encompassed in the scope of the present disclosure as set forth in the appended claims.

Claims

CMUH-0002PCTUS CLAIMS What is claimed is:

1. A method for preventing or treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a composition comprising an agent having a serine kinase inhibition activity against human epidermal growth factor receptor 2 (HER2) and a pharmaceutically acceptable carrier thereof.

2. The method of claim 1, wherein the serine kinase inhibition activity is against at least one of serine 1051 and serine 1054 of HER2.

3. The method of claim 1, wherein the agent further has a tyrosine kinase inhibition activity against HER2.

4. The method of claim 1, wherein the subject has increased expression or activation of HER2.

5. The method of claim 1, wherein the subject is previously treated with an anti-cancer therapeutic agent.

6. The method of claim 5, wherein the subject develops resistance to the anti-cancer therapeutic agent.

7. The method of claim 5, wherein the anti-cancer therapeutic agent is at least one of trastuzumab, pertuzumab, trastuzumab emtansine, trastuzumab deruxtecan, lapatinib, neratinib, pyrotinib, and tucatinib.

8. The method of claim 1, wherein the subject has refractory cancer.

9. The method of claim 1, wherein the cancer is colorectal cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, head and neck cancer, pancreatic cancer, non-small cell lung cancer, glioblastoma, bladder cancer, or cervical cancer.

10. The method of claim 9, wherein the cancer is gastric cancer or breast cancer.

11. The method of claim 9, wherein the cancer has a brain metastasis.

12. The method of claim 1, wherein the agent has a structure ofCMUH-0002PCTUS13. A method for preventing or treating cancer in a subject in need thereof, comprising inhibiting a serine kinase activity of human epidermal growth factor receptor 2 (HER2).

14. The method of claim 13, wherein at least one of serine 1051 and serine 1054 of the serine kinase activity of HER2 is inhibited.

15. The method of claim 13, further comprising inhibiting a tyrosine kinase activity of HER2.

16. A method for identifying an agent for preventing or treating cancer in a subject in need thereof, comprising screening for a compound having a kinase inhibition activity against at least one of serine 1051 and serine 1054 of human epidermal growth factor receptor 2 (HER2).

17. The method of 16, further comprising screening for a compound having a kinase inhibition activity against a tyrosine of HER2.CMUH-0002PCTUS 18. The method of 17, wherein the tyrosine is at least one of Tyr877, Tyr1005, Tyr1023, Tyr1112, Tyr1139, Tyr1196, Tyr1221, Tyr1222, and Tyr1248 of HER2.

19. A method for preventing or treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a composition comprising an agent identified by any one of the methods of claims 16 to 18.

20. A pharmaceutical composition for use in preventing or treating cancer in a subject in need thereof, the pharmaceutical composition comprising an agent having a serine kinase inhibition activity against human epidermal growth factor receptor 2 (HER2) and a pharmaceutically acceptable carrier thereof.

21. The pharmaceutical composition for use of claim 20, wherein the serine kinase inhibition activity is against at least one of serine 1051 and serine 1054 of HER2.

22. The pharmaceutical composition for use of claim 20, wherein the agent further has a tyrosine kinase inhibition activity against HER2.

23. The pharmaceutical composition for use of claim 20, wherein the subject has increased expression or activation of HER2.

24. The pharmaceutical composition for use of claim 20, wherein the subject is previously treated with an anti-cancer therapeutic agent.

25. The pharmaceutical composition for use of claim 24, wherein the subject develops resistance to the anti-cancer therapeutic agent.

26. The pharmaceutical composition for use of claim 25, wherein the anti-cancer therapeutic agent is at least one of trastuzumab, pertuzumab, trastuzumab emtansine, trastuzumab deruxtecan, lapatinib, neratinib, pyrotinib, and tucatinib.

27. The pharmaceutical composition for use of claim 20, wherein the subject has refractory cancer.

28. The pharmaceutical composition for use of claim 20, wherein the cancer is colorectal cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, head and neck cancer, pancreatic cancer, non-small cell lung cancer, glioblastoma, bladder cancer, or cervical cancer.

29. The pharmaceutical composition for use of claim 28, wherein the cancer is gastric cancer or breastCMUH-0002PCTUS cancer.

30. The pharmaceutical composition for use of claim 28, wherein the cancer has a brain metastasis.

31. The pharmaceutical composition for use of claim 20, wherein the agent has a structure of32. A pharmaceutical composition for use in preventing or treating cancer in a subject in need thereof, the pharmaceutical composition comprising an agent identified by screening for a kinase inhibition activity against at least one of serine 1051 and serine 1054 of human epidermal growth factor receptor 2 (HER2) and a pharmaceutically acceptable carrier thereof.

Images