Drugs and their uses for targeted therapy of CDK4 / 6 inhibitor-resistant breast cancer

Abstract

This invention relates to pharmaceutical compositions targeting HER2 for the treatment of CDK4 / 6 inhibitor-resistant breast cancer and their applications. Administering a therapeutically effective amount of a HER2 inhibitor can reverse or prevent breast cancer from developing resistance to CDK4 / 6 inhibitors. This invention proposes a new paradigm for reversing resistance by targeting HER2 and demonstrates a strong synergistic effect when CDK4 / 6 inhibitors are used in combination with HER2-targeting inhibitors, providing a novel direction for addressing treatment after CDK4 / 6 inhibitor resistance develops.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of HER2 inhibitors in the preparation of drugs for treating estrogen receptor-positive breast cancer resistant to CDK4 / 6 inhibitors, and the pharmaceutical composition or kit thereof. Background Technology

[0002] Breast cancer is the most common malignant tumor among women, with the hormone receptor (HR) positive, human epidermal growth factor receptor 2 (HER2) negative subtype accounting for approximately 65%-70% of all breast cancers, making it the most common molecular subtype. For this subtype, endocrine therapy (such as tamoxifen and aromatase inhibitors) has long been the first-line standard treatment. However, the widespread presence of primary and acquired resistance severely limits the clinical benefit of endocrine therapy; more than 50% of HR+ metastatic breast cancer patients develop resistance to endocrine therapy, making disease progression inevitable.

[0003] Cyclin-dependent kinases 4 and 6 (CDK4 / 6) are key regulators of the cell cycle transition from G1 to S phase. They phosphorylate retinoblastoma protein (Rb) by forming a complex with Cyclin D, releasing the E2F transcription factor and initiating DNA replication. In HR+ breast cancer, aberrant activation of the CDK4 / 6-Cyclin D-Rb pathway is one of the important mechanisms of endocrine therapy resistance. Based on this understanding, CDK4 / 6 inhibitors have emerged, representing the most groundbreaking advancement in the treatment of HR+ breast cancer in the past decade.

[0004] CDK4 / 6 inhibitors such as abemaciclib, palbociclib, and ribociclib have significantly improved the survival of patients with hormone receptor-positive breast cancer, but acquired resistance leads to treatment failure in approximately 30% of cases (Finnet al., N Engl J Med 2016; 375:1925-1936). Therefore, exploring the resistance characteristics of CDK4 / 6 inhibitors and finding new treatment options is of great clinical significance and scientific value.

[0005] Current research on CDK4 / 6 inhibitor resistance mechanisms focuses on: Firstly, acquired mutations in estrogen receptor α (ESR1) activate downstream MAPK / PI3K bypass signaling: the ER signaling pathway is the mainstay of survival in HR+ breast cancer. Under the dual pressure of CDK4 / 6 inhibitors and endocrine therapy, tumor cells can achieve ligand-independent activation of the ER through acquired mutations in the ESR1 gene (most commonly Y537S and D538G), continuously driving downstream transcriptional programs even in the absence of estrogen. These mutant ERs not only directly upregulate cyclin D1 to attempt to "counteract" the inhibitory effect of CDK4 / 6, but also activate bypass survival signals such as MAPK / ERK and PI3K / AKT / mTOR through genome-wide chromatin remodeling, forming a cross-escape from CDK4 / 6 inhibitors. Secondly, cell cycle checkpoint escape: CDK4 / 6 inhibitors target the G1 / S phase checkpoint. When this checkpoint is effectively blocked, resistant cells can restart the cell cycle through various means, such as Rb protein inactivation, i.e., truncated mutations or deletions of the RB1 gene leading to complete Rb inactivation, thus rendering the inhibitory effect of the CDK4 / 6-Rb axis completely ineffective, and the cell cycle process no longer regulated by CDK4 / 6; compensatory upregulation of the CDK2-cyclin E complex: resistant cells often activate the CDK2-cyclin E pathway by upregulating cyclin E1 (CCNE1) expression or reducing the level of endogenous CDK2 inhibitors (such as p27^Kip1), bypassing the inhibited CDK4 / 6 to directly phosphorylate Rb and drive the cell cycle.

[0006] Despite significant progress in understanding the aforementioned resistance mechanisms, their clinical translational value remains fundamentally limited. First, ESR1 mutation-targeting drugs (such as novel oral SERD agents) and PI3K / AKT pathway inhibitors (such as Alpelisib and Capivasertib) offer limited clinical benefit in later-line treatment, with objective response rates typically only 10%–20%, and are constrained by cumulative toxicity and tolerability issues. Second, CDK2 inhibitors are still in early clinical stages, exhibiting limited single-agent activity and facing safety challenges due to poor binding selectivity among CDK family members.

[0007] Clinical management strategies have fundamental limitations: after drug resistance develops, there is a lack of targeted intervention options, and most patients turn to cytotoxic chemotherapy (5-year survival rate <40%). An even more prominent contradiction lies in the fact that traditional classification considers ER... + Most breast cancers are HER2-negative. Clinical studies have shown that the HER2 inhibitor lapatinib has a positive effect on HER2-negative breast cancer. + / HER2 -The objective response rate in patients was only 5.9% (Johnston et al., JClin Oncol 2013; 31:1955-1962), which led to the long-term neglect of the role of the HER2 pathway in CDK4 / 6 inhibitor resistance. Furthermore, there has never been evidence that CDK4 / 6 inhibitors can directly regulate HER2 expression, directly resulting in the prevailing view that the HER2 pathway has no key driving role in this subtype, thus excluding HER2 from the mainstream framework of resistance mechanism research. However, the above conclusions were drawn under baseline conditions lacking CDK4 / 6 inhibitor treatment pressure. Whether tumor cells gain a survival advantage under the selection pressure created by long-term exposure to CDK4 / 6 inhibitors through non-genetic mutation methods (such as transcriptional reprogramming and epigenetic remodeling) by upregulating HER2 or its downstream effector molecules (such as p-HER2, p-MAPK, and p-AKT) remains a question that has not been systematically reported to date. More importantly, there is currently no evidence that CDK4 / 6 inhibitors can directly or indirectly regulate the expression level or phosphorylation activation state of HER2—neither their regulatory effect has been confirmed nor their possibility ruled out. The role of the HER2 pathway in the CDK4 / 6 inhibitor resistance landscape thus constitutes a long-neglected knowledge gap.

[0008] In summary, how to systematically analyze the molecular regulatory relationship between CDK4 / 6 inhibitors and HER2, clarify whether the HER2 signaling pathway constitutes a functional driving factor for CDK4 / 6 inhibitor resistance, and on this basis, provide a targeted therapy drug for CDK4 / 6 inhibitor-resistant breast cancer with HER2 as the target and its pharmaceutical applications, is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0009] The first objective of this invention is to address the problems in the prior art by providing the use of a HER2 inhibitor in the preparation of a medicament for treating CDK4 / 6 inhibitor-resistant breast cancer.

[0010] This invention provides the use of a HER2 inhibitor in the preparation of a medicament for treating CDK4 / 6 inhibitor-resistant breast cancer, wherein the HER2 inhibitor is selected from at least one or a combination of neratinib, afatinib, gefitinib, lapatinib, tucatinib, T-DM1, or trastuzumab.

[0011] In another embodiment, the present invention provides the use of a HER2 inhibitor in the preparation of a medicament for treating CDK4 / 6 inhibitor-resistant breast cancer, wherein the HER2 inhibitor is selected from at least one or a combination of neratinib, afatinib, or gefitinib.

[0012] In another embodiment, the present invention provides the use of a HER2 inhibitor in the preparation of a medicament for treating CDK4 / 6 inhibitor-resistant breast cancer, wherein the HER2 inhibitor is neratinib.

[0013] In another embodiment, the present invention provides the use of a HER2 inhibitor in the preparation of a medicament for treating CDK4 / 6 inhibitor-resistant breast cancer, wherein the CDK4 / 6 inhibitor that causes resistance in breast cancer is at least one or a combination of abecicilline, pebocilline, ribociclib, dalcilib, or triplaccilline.

[0014] In another embodiment, the present invention provides the use of a HER2 inhibitor in the preparation of a medicament for treating CDK4 / 6 inhibitor-resistant breast cancer, wherein the CDK4 / 6 inhibitor that causes resistance in breast cancer is abexicillin, pebocillin, or ribociclib.

[0015] In another embodiment, the present invention provides the use of a HER2 inhibitor in the preparation of a medicament for treating CDK4 / 6 inhibitor-resistant breast cancer, wherein the breast cancer is estrogen receptor-positive and HER2-negative breast cancer.

[0016] A second objective of the present invention is to provide a pharmaceutical composition that can be used to reverse or prevent resistance to CDK4 / 6 inhibitors in breast cancer patients.

[0017] The present invention provides a pharmaceutical composition that can be used to reverse or prevent resistance to CDK4 / 6 inhibitors in breast cancer patients, wherein the pharmaceutical composition comprises a therapeutically effective amount of a HER2 inhibitor and a CDK4 / 6 inhibitor, wherein the HER2 inhibitor is selected from at least one of neratinib, afatinib, gefitinib, lapatinib, tucatinib, T-DM1, or trastuzumab, and wherein the CDK4 / 6 inhibitor is selected from at least one of abeciclib, pebocilib, ribociclib, dalcilib, or trelacidlib.

[0018] In another embodiment, the present invention provides a pharmaceutical composition that can be used to reverse or prevent resistance to CDK4 / 6 inhibitors in breast cancer patients, wherein the pharmaceutical composition or kit comprises a therapeutically effective amount of a HER2 inhibitor and a CDK4 / 6 inhibitor, wherein the HER2 inhibitor is selected from neratinib, afatinib, or gefitinib, and wherein the CDK4 / 6 inhibitor is selected from abeciclib, pebocilib, or ribociclib.

[0019] In another preferred embodiment, the present invention provides a pharmaceutical composition that can be used to reverse or prevent resistance to CDK4 / 6 inhibitors in breast cancer patients, wherein the pharmaceutical composition or kit contains therapeutically effective amounts of a HER2 inhibitor and a CDK4 / 6 inhibitor, wherein the HER2 inhibitor is neratinib, afatinib, or gefitinib, and wherein the CDK4 / 6 inhibitor is abexilide.

[0020] In another preferred embodiment, the present invention provides a pharmaceutical composition that can be used to reverse or prevent resistance to CDK4 / 6 inhibitors in breast cancer patients, wherein the breast cancer is estrogen receptor-positive and HER2-negative breast cancer.

[0021] A third objective of this invention is to provide a kit that can be used to reverse or prevent resistance to CDK4 / 6 inhibitors in breast cancer patients.

[0022] This invention provides a kit for reversing or preventing resistance to CDK4 / 6 inhibitors in breast cancer patients, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from at least one of neratinib, afatinib, gefitinib, lapatinib, tucatinib, T-DM1, or trastuzumab; and (2) Individually packaged CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitor is selected from at least one of abecicilline, pebocilline, ribocicline, dalcicilline or trilaccilline.

[0023] In another embodiment, the present invention provides a kit for reversing or preventing resistance to CDK4 / 6 inhibitors in breast cancer patients, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from neratinib, afatinib, or gefitinib; and (2) Individually packaged CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitors are selected from abecicilline, pebocilline or ribociclib.

[0024] In another embodiment, the present invention provides a kit for reversing or preventing resistance to CDK4 / 6 inhibitors in breast cancer patients, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from neratinib, afatinib, or gefitinib; and (2) Individually packaged CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitor is abecilibi.

[0025] In another preferred embodiment, the present invention provides a kit that can be used to reverse or prevent resistance to CDK4 / 6 inhibitors in breast cancer patients, wherein the breast cancer is estrogen receptor-positive and HER2-negative breast cancer.

[0026] In another preferred embodiment, the present invention provides a kit for reversing or preventing resistance to CDK4 / 6 inhibitors in estrogen receptor-positive and HER2-negative breast cancer patients, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from at least one of neratinib, afatinib, gefitinib, lapatinib, tucatinib, T-DM1, or trastuzumab; and (2) Individually packaged CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitor is selected from at least one of abecicilline, pebocilline, ribociclib, dalcilib, or trilaccilline; The method of using the kit is as follows: first, administer a CDK4 / 6 inhibitor to the patient, and after the patient develops resistance, treat with a HER2 inhibitor to restore the sensitivity of the drug-resistant cells to the CDK4 / 6 inhibitor.

[0027] In another preferred embodiment, the present invention provides a kit for reversing or preventing resistance to CDK4 / 6 inhibitors in estrogen receptor-positive and HER2-negative breast cancer patients, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from neratinib, afatinib, or gefitinib; and (2) Individually packaged CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitor is selected from abecicilline, pebocilline or ribociclib; The method of using the kit is as follows: first, administer a CDK4 / 6 inhibitor to the patient, and after the patient develops resistance, treat with a HER2 inhibitor to restore the sensitivity of the drug-resistant cells to the CDK4 / 6 inhibitor.

[0028] In another preferred embodiment, the present invention provides a kit for reversing or preventing resistance to CDK4 / 6 inhibitors in estrogen receptor-positive and HER2-negative breast cancer patients, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from neratinib, afatinib, or gefitinib; and (2) Individually packaged CDK4 / 6 inhibitor abexib; The method of using the kit is as follows: first, administer the CDK4 / 6 inhibitor abexib to the patient, and after the patient develops resistance, treat the patient with the HER2 inhibitor to restore the sensitivity of the resistant cells to the CDK4 / 6 inhibitor.

[0029] In another preferred embodiment, the present invention provides a kit for reversing or preventing resistance to CDK4 / 6 inhibitors in estrogen receptor-positive and HER2-negative breast cancer patients, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from at least one of neratinib, afatinib, gefitinib, lapatinib, tucatinib, T-DM1, or trastuzumab; and (2) Individually packaged CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitor is selected from at least one of abecicilline, pebocilline, ribociclib, dalcilib, or trilaccilline; The method of using the kit is as follows: first, administer CDK4 / 6 inhibitors to the patient; after the patient develops resistance, administer HER2 inhibitors to restore the sensitivity of the resistant cells to CDK4 / 6 inhibitors; and then administer CDK4 / 6 inhibitors to the patient again.

[0030] In another preferred embodiment, the present invention provides a kit for reversing or preventing resistance to CDK4 / 6 inhibitors in estrogen receptor-positive and HER2-negative breast cancer patients, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from neratinib, afatinib, or gefitinib; and (2) Individually packaged CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitor is selected from abecicilline, pebocilline or ribociclib; The method of using the kit is as follows: first, administer CDK4 / 6 inhibitors to the patient; after the patient develops resistance, administer HER2 inhibitors to restore the sensitivity of the resistant cells to CDK4 / 6 inhibitors; and then administer CDK4 / 6 inhibitors to the patient again.

[0031] In another preferred embodiment, the present invention provides a kit for reversing or preventing resistance to CDK4 / 6 inhibitors in estrogen receptor-positive and HER2-negative breast cancer patients, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from neratinib, afatinib, or gefitinib; and (2) Individually packaged CDK4 / 6 inhibitor abexib; The method of using the kit is as follows: first, administer the CDK4 / 6 inhibitor abexib to the patient; after the patient develops resistance, treat the patient with the HER2 inhibitor to restore the sensitivity of the resistant cells to the CDK4 / 6 inhibitor; and then administer the CDK4 / 6 inhibitor to the patient again.

[0032] This invention unexpectedly discovered that exposure to CDK4 / 6 inhibitors continuously activates the HER2 pathway. Cells that have developed resistance exhibit significantly increased sensitivity to HER2 inhibitors. Treatment with HER2 inhibitors at this point effectively reverses resistance and restores cellular sensitivity to CDK4 / 6 inhibitors. This invention demonstrates that HER2 upregulation through HER2 inhibitor administration is not only related to CDK4 / 6 inhibitor resistance, but that CDK4 / 6 inhibitor-induced HER2 expression upregulation is a key mechanism leading to acquired resistance. Therefore, inhibiting HER2 is an effective means of overcoming and reversing CDK4 / 6 inhibitor resistance in breast cancer cells, and the combination of HER2 and CDK4 / 6 inhibitors produces a significant synergistic killing effect.

[0033] For example, after 8 weeks of treatment with the CDK4 / 6 inhibitor abexicillin, ER+ breast cancer cells MCF-7 and T47D showed a significant increase in HER2 mRNA levels. In the drug-resistant cell line (MCF-7RA / T47DRA), the basal level of HER2 protein was significantly increased compared with the parental cells, accompanied by enhanced malignant phenotypes, including stronger 3D clonogenic ability and in vivo tumorigenesis ability. Furthermore, the drug-resistant cells showed a significant increase in sensitivity to second-generation HER2 inhibitors.

[0034] Based on this, this invention proposes a new paradigm for reversing drug resistance by targeting HER2, and demonstrates that the combination of CDK4 / 6 inhibitors and HER2-targeting inhibitors (in drug composition or kit form) produces a strong synergistic effect, with a peak SynergyScore of 8.6 in the ZIP model, providing a novel direction for treating CDK4 / 6 inhibitor resistance. In clinical practice, for example, estrogen receptor-positive and HER2-negative breast cancer patients can first be given CDK4 / 6 inhibitors, and after resistance develops, HER2 inhibitors can be administered to restore the sensitivity of resistant cells to CDK4 / 6 inhibitors. Then, CDK4 / 6 inhibitors can be administered to the patients again to improve clinical treatment efficacy. The dosing regimens for CDK4 / 6 inhibitors and HER2 inhibitors can be followed according to the dosing regimens specified in their respective approved drug instructions. Attached Figure Description

[0035] Figure 1 Treatment of MCF-7 and T47D cells with three CDK4 / 6 inhibitors significantly increased HER2 mRNA and protein levels. A represents the results for MCF-7 cells, and B represents the results for T47D cells.

[0036] Figure 2 The results showed that the increase in HER2 levels induced by abexicillin in MCF-7 and T47D cells was time- and concentration-dependent. In A, HER2 mRNA levels in MCF-7 and T47D cells gradually increased with prolonged abexicillin treatment; in B, HER2 protein levels in MCF-7 and T47D cells gradually increased with increasing abexicillin concentration.

[0037] Figure 3 The results show that the HER2 protein level was significantly elevated in abexicillin-resistant cell lines MCF-7 and T47D. A shows the results of western blot analysis, indicating a significant increase in HER2 protein levels in abexicillin-resistant cell lines MCF-7 and T47D; B shows the results of immunofluorescence staining, indicating a significant increase in HER2 protein levels in abexicillin-resistant cell line MCF-7.

[0038] Figure 4 The results show the enhanced 3D colony-forming ability of MCF-7RA and T47DRA cells. A represents the experimental results for MCF-7RA cells, and B represents the experimental results for T47DRA cells.

[0039] Figure 5 The results show the sensitivity of abexicillin-resistant cell lines MCF-7 and T47D (MCF-7RA, T47DRA) to the HER2 inhibitors afatinib, gefitinib, and neratinib under 2D culture conditions. In the figures, A represents the experimental results for MCF-7RA cells, and B represents the experimental results for T47DRA cells.

[0040] Figure 6 The results show that MCF-7RA and T47DRA cells are more sensitive to three HER2 inhibitors (Afatinib, Gefitinib, and Neratinib) under 3D culture conditions. A represents the experimental results for MCF-7RA cells, and B represents the experimental results for T47DRA cells.

[0041] Figure 7 The results indicate a synergistic effect when three HER2 inhibitors (Afatinib, Gefitinb, Neratinib) are used in combination with Abemaciclib.

[0042] Figure 8 The results show the restoration of sensitivity to abemaciclib in MCF-7 abemaciclib-resistant cells (MCF-7RA) after treatment with neratinib. A represents CCK8 staining results after 2 days of culture, and B represents AO / PI staining results after 3 days of culture.

[0043] Figure 9 This indicates that Neratinib can inhibit the progression of MCF-7RA xenografts. A shows the tumor growth curves for MCF-7 and MCF-7RA; B shows tumor images for each group; and C shows the tumor weight for each group. Detailed Implementation

[0044] The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

[0045] This invention reveals a novel targeted therapy strategy for CDK4 / 6 inhibitor-resistant breast cancer, the core of which lies in discovering and utilizing the key resistance phenomenon of abnormal activation of the HER2 pathway. Specifically, it includes: 1. Mechanism discovery: CDK4 / 6 inhibitor treatment directly induces ER + HER2 expression levels were significantly increased in breast cancer cells (MCF-7 / T47D) (time-dependent: mRNA increased more than 10-fold after 8 weeks; concentration-dependent: positive correlation between 0-400 nM). HER2 protein was persistently highly expressed in the drug-resistant strain (MCF-7RA / T47DRA) and the malignant phenotype was enhanced (3D clonogenic ability and in vivo tumor growth were enhanced). 2. HER2-targeting efficacy: In vitro experiments demonstrated that, under 2D and 3D culture conditions, drug-resistant cells MCF-7RA and T47DRA showed significantly increased sensitivity to HER2 inhibitors Afatinib, Gefitinib, and Neratinib compared to parental cells. In vivo experiments demonstrated that Neratinib had no significant inhibitory effect on the growth of normal MCF-7 xenografts, while it achieved an 83% inhibition rate on the growth of drug-resistant MCF-7RA xenografts.

[0046] 3. Synergistic Treatment Regimen: Validation using a concentration combination matrix and ZIP model showed that abexicillin combined with a HER2 inhibitor produced a strong synergistic effect (Synergy Score > 5, with a peak score of 8.6 for the Neratinib combination). This combination significantly enhanced the inhibitory effect on CDK4 / 6 inhibitor-resistant breast cancer cells.

[0047] 4. HER2 Inhibitor Reversal of CDK4 / 6 Inhibitor Resistance: Pretreatment of CDK4 / 6 inhibitor-resistant cells with HER2 inhibitors can significantly restore the sensitivity of resistant cells to CDK4 / 6 inhibitors (Neratinib pretreatment increases the sensitivity of MCF-7RA to abexicillin, IC50...). 50 (Decreased from 0.54 μM to 0.2 μM).

[0048] The technical solution of this application embodiment is to solve the above-mentioned technical problems, and the general idea is as follows: The first part shows that CDK4 / 6 inhibitors significantly upregulated HER2 levels (mRNA and protein) in MCF-7 and T47D cells in a time- and concentration-dependent manner, and the induced abexicillin-resistant cells (MCF-7RA, T47DRA) showed sustained high HER2 expression and enhanced 3D cloning ability.

[0049] The second part is about the sensitivity of abexicillin-resistant cells to HER2 inhibitors (in vitro and in vivo); the synergistic effect of HER2 inhibitors combined with CDK4 / 6 inhibitors; and the ability of HER2 inhibitor pretreatment to reverse resistance and restore sensitivity to abexicillin.

[0050] Example 1: Verification of CDK4 / 6 inhibitor-induced upregulation of HER2 expression 1. Cell Culture and Drug Treatment (1) Human ER+ breast cancer cell lines MCF-7 and T47D were maintained using standard culture conditions.

[0051] (2) HER2 mRNA level detection (RT-qPCR): Cells were seeded in six-well plates and treated with 100 nM abecicilline, pebocilline, and ribociclib for 48 h after adhesion. Cells were harvested, total RNA was extracted, and reverse transcribed into cDNA. Real-time quantitative PCR (RT-qPCR) of the HER2 gene was performed using specific primers. The internal reference gene GAPDH was used for normalization. The relative expression level of HER2 mRNA was calculated (ΔΔCt method), and the changes in HER2 mRNA levels after treatment with the three CDK4 / 6 inhibitors were analyzed.

[0052] HER2 qPCR primer sequences: Leader chain 5'- TGCAGGGAAACCTGGAACTC-3'; Followed chain 5'-ACAGGGGTGGTATTGTTCAGC-3'.

[0053] (3) HER2 protein level detection: Similar to the mRNA experiment setup, cells were treated with 100 nM of three CDK4 / 6 inhibitors for 48 h. Cells were harvested, lysed, and total protein was extracted and quantified. SDS-PAGE electrophoresis was performed, and the cells were transferred to a PVDF membrane. The cells were incubated with a specific anti-HER2 primary antibody, followed by incubation with the corresponding secondary antibody. Electrochemiluminescence was used to develop and detect HER2 protein bands. The internal control protein GAPDH was used for sample loading standardization. By analyzing the gray values, the changes in HER2 protein expression levels were quantified, and it was found that the protein levels of MCF-7 and T47D were significantly increased after treatment with CDK4 / 6 inhibitors.

[0054] The results are as follows Figure 1As shown, treatment of MCF-7 and T47D cells with abecili, pebocilli, and ribociclib significantly increased the mRNA and protein levels of HER2.

[0055] 2. Time- and concentration-dependent increase in HER2 expression levels by abecicilline (1) Maintaining human ER using standard culture conditions + Breast cancer cell lines MCF-7 and T47D.

[0056] (2) Investigation on the correlation between HER2 expression level and abexilide induction time: To investigate the effects of long-term abexicillin induction on HER2 expression, we collected cell samples at different time points (2, 3, 4, 8, and 12 weeks) during an induction period ranging from 2 to 12 weeks. Total RNA was extracted and reverse transcribed into cDNA. HER2 mRNA expression levels were detected by real-time quantitative PCR (RT-qPCR), with GAPDH as an internal reference gene. The relative expression level was calculated using the ΔΔCt method to analyze the trend of HER2 transcriptional levels with induction time.

[0057] (3) HER2 expression level and its dependence on abexilide concentration: To analyze the concentration-dependent effect of abexicillin treatment on HER2 protein expression, MCF-7 and T47D parental cells were exposed to abexicillin at concentration gradients of 0–400 nM for 48 hours. Cellular protein samples were collected after treatment, and protein quantification was performed using the BCA method. After SDS-PAGE electrophoresis, membrane transfer, and blocking, the cells were incubated with a specific HER2 primary antibody and a GAPDH internal control antibody, followed by ECL chemiluminescence imaging. Changes in HER2 protein expression with varying drug concentrations were assessed by analyzing the grayscale values ​​of the protein bands.

[0058] The results are as follows Figure 2 As shown, with prolonged induction time (from 2 to 12 weeks), the mRNA expression level of HER2 in MCF-7 and T47D cells exhibited time-dependent upregulation. Treatment with different concentration gradients of abexicillin (0-400 nM) for 48 hours resulted in increased HER2 protein expression with increasing drug concentration, indicating that abexicillin treatment itself can induce concentration-dependent activation of HER2 expression in the short term. These results collectively suggest that HER2 upregulation is not only a potential marker of long-term acquired drug resistance but also an early response to acute drug stress.

[0059] Example 2 Construction of abexicillin-resistant cell lines 1. Establishment and validation of abexicillin-resistant cell lines (MCF-7RA, T47DRA) (1) Drug resistance induction: Parental MCF-7 and T47D cells were continuously exposed to an increasing concentration of abexicillin (starting at 50 nM) for 3 months. Cell viability was monitored regularly during this period, and the drug concentration was gradually increased to screen out cell populations that could stably proliferate under high concentrations of abexicillin (drug-resistant strains, named MCF-7RA and T47DRA).

[0060] (2) Drug resistance confirmation: The survival rates of parental cells and drug-resistant cells under different concentrations of abexicillin were compared by using the CCK8 cell viability assay. The dose-response curve was plotted, the IC50 value was calculated, and the drug resistance of the cells was confirmed.

[0061] (3) Detection of HER2 protein expression in drug-resistant cells (Western Blot): Parental cells and drug-resistant cells were cultured to the logarithmic growth phase under drug-free conditions, and the protein was harvested. HER2 expression was detected by Western Blot (method as above) to confirm the sustained high expression of HER2 protein in drug-resistant cells.

[0062] The results are as follows Figure 3 As shown, the expression levels of HER2 protein in abexicillin-resistant cells MCF-7RA and T47DRA were compared with their corresponding parental cells MCF-7 and T47D using Western blotting (WB) and immunofluorescence assays. WB results showed that HER2 protein expression in both MCF-7RA and T47DRA cells was significantly higher than in their control cells. Immunofluorescence images further confirmed that the HER2 signal (green fluorescence) in MCF-7RA cells was significantly stronger than that in the MCF-7 control group, suggesting that HER2 is significantly upregulated in abexicillin-resistant cells.

[0063] 2.3D Clonal Formation (Evaluation of Stem Cell-like Characteristics) Cell pretreatment: Collect parental cells (MCF-7 / T47D) and drug-resistant cells (MCF-7RA / T47DRA), centrifuge at 1200 r / min for 5 min, and discard the supernatant. Resuspend the cell pellet in matrix gel on ice. Cell density: 2000 cells / 5 μL. Matrix gel concentration: 100% (without medium dilution). Drop 5 μL of the cell-matrix gel mixture into a 96-well plate to form a hanging drop, and invert the plate to incubate at 37°C. Incubate for 15–20 minutes to allow the hanging drop to solidify into a hemispherical gel. Culture medium addition and culture: Add 100 μL of 3D complete medium (formulation: DMEM / F12 + 50 ng / mL EGF + 1× ITS + 1× P / S) to each well of the plate. Carefully replace the cap of the plate with the solidified hanging drop, ensuring the droplet is submerged in the medium. Change the medium every 2 days and culture for 8 days. By taking photos and analyzing the results, the size and number of cell spheres from 3D culture were obtained.

[0064] The results are as follows Figure 4 As shown, after 8 days of culture in 3D matrix gel, the 3D clones formed by abexicillin-resistant cells MCF-7RA and T47DRA were significantly larger than their parent cells, indicating that the 3D clone-forming ability of abexicillin-resistant cells was significantly enhanced.

[0065] Example 3: Verification of the sensitivity and synergistic effect of CDK4 / 6 inhibitor-resistant cells to HER2 inhibitors. 1. HER2 inhibitors significantly inhibited the activity of MCF-7RA cells in vitro. 1.1 Inhibition of MCF-7RA cell activity by HER2 inhibitors under 2D culture conditions To evaluate the in vitro inhibitory effect of HER2 inhibitors on abexicillin-resistant cells, we first performed a two-dimensional (2D) cell viability assay. MCF-7RA cells and their parental MCF-7 cells were seeded at a density of 3000 cells per well in 96-well plates and cultured for 24 hours. Then, the cells were treated with gradients of afatinib, gefitinib, and neratinib for 48 hours, respectively. Cell viability was then assessed using the CCK-8 assay, measuring absorbance at 450 nm and calculating the IC50 of each drug by fitting dose-response curves using GraphPad Prsm software. 50 value.

[0066] The results are as follows Figure 5 As shown, the sensitivity of MCF-7RA and T47DRA cells to three HER2 inhibitors (Afatinib, Gefitinib, and Neratinib) was evaluated under two-dimensional culture conditions using the CCK-8 assay. The results showed that, compared with their corresponding parental cells, the IC50 values ​​of MCF-7RA and T47DRA cells to each inhibitor were significantly higher. 50 The values ​​were all significantly reduced, indicating that abecilibi-resistant cells are more sensitive to HER2 inhibitors.

[0067] 1.2 Killing effect of HER2 inhibitors on drug-resistant cell spheroids in 3D culture model To further validate our findings in a model that more closely resembles the in vivo environment, we employed a three-dimensional (3D) culture system. MCF-7, MCF-7RA, T47D, and T47DRA cells (2000 cells per drop in 5 μL of Matrigel) were cultured to form spheroids for 8 days. After spheroid maturation, the cells were treated with 500 nM of the three HER2 inhibitors for 48 hours. Following treatment, acridine orange (AO) / propidium iodide (PI) double staining was performed, followed by incubation in the dark for 30 minutes. Image capture and analysis were then conducted to assess the viability and mortality of the spheroids.

[0068] The results are as follows Figure 5As shown, under three-dimensional culture conditions, AO / PI staining revealed that MCF-7RA cells underwent significant apoptosis after treatment with three HER2 inhibitors, while the MCF-7 parental cells showed no significant response; the same trend was also observed between the T47DRA and T47D cell groups, further confirming that drug-resistant cells have higher sensitivity to HER2 inhibitors in the 3D model.

[0069] 2. Abecilibaly has a synergistic effect with all three HER2 inhibitors. Drug-resistant MCF-7RA / T47DRA cells were seeded in 96-well plates. After 24 hours, three independent experiments were performed (abecidil combined with afatinib, gefitinib, or neratinib, respectively). Each group included: ① abexicillin monotherapy concentration gradient; ② HER2 inhibitor monotherapy concentration gradient; ③ a fixed molar ratio combination matrix of two drugs (6×6 concentration gradients). After 72 hours of treatment, cell death was assessed using the CCK-8 assay. The drug concentration-mortality matrix data from the three experiments were imported into the SynergyFinder website (https: / / synergyfinder.fimm.fi / ), and a ZIP (Zero Interaction Potency) model was selected for synergistic effect analysis. Results were quantified: a synergistic score was output, and the combined effect strength was visualized using a heatmap, confirming a synergistic effect between CDK4 / 6 inhibitors and HER2 inhibitors.

[0070] The results are as follows Figure 7 As shown in the figure. ZIP model analysis showed that abecitabine combined with each HER2 inhibitor exhibited a significant synergistic effect, with synergistic scores of 5.65, 6.26 and 8.6, respectively, and all combinations were statistically significant (p < 0.001), with the combination of abecitabine and neratinib showing the strongest synergistic effect.

[0071] This demonstrates that (1) drug-resistant cells are significantly more sensitive to HER2 inhibitors; (2) the combination of HER2 inhibitors and CDK4 / 6 inhibitors produces a significant synergistic killing effect; and (3) HER2 inhibitor pretreatment can effectively reverse drug resistance and restore cell sensitivity to CDK4 / 6 inhibitors. This proves that HER2 upregulation through HER2 inhibitor administration is not only related to CDK4 / 6 inhibitor resistance, but that CDK4 / 6 inhibitor-induced HER2 upregulation is a key mechanism leading to acquired resistance. Therefore, inhibiting HER2 is an effective means to overcome and reverse resistance to CDK4 / 6 inhibitors in breast cancer cells.

[0072] 3. Neratinib can reverse the resistance of MCF-7RA to abexicillin. MCF-7RA resistant cells were treated with 100 nM Neratinib for 48 hours (cell viability >90%), and the cells were collected after complete drug elution. 2D validation: Pretreated cells and untreated control cells were seeded in 96-well plates and treated with abexicillin at gradient concentrations (25-3200 nM) for 48 hours. Viability was assessed using the CCK-8 assay. 3D validation: Cells pretreated in the same manner were used to construct 3D spheroids (2000 cells / 5 μL Matrigel), cultured for 4 days, and then treated with the same gradient of abexicillin (25-3200 nM) for 48 hours. Endpoint detection: AO / PI double staining (incubation in the dark for 30 minutes) was performed to analyze spheroid viability.

[0073] The results are as follows Figure 8 As shown, after pretreatment with 100 nM Neratinib for 48 hours, MCF-7RA cells showed a significant increase in sensitivity to abexicillin, with an IC50 value of [missing information]. 50 The value decreased from 0.54 μM to 0.2 μM. Simultaneously, under 3D culture conditions, AO / PI experiments also verified that Neratinib treatment could restore the sensitivity of MCF-7RA cells to abexicillin. These results collectively demonstrate that the HER2 inhibitor Neratinib can effectively reverse the resistance of MCF-7RA cells to abexicillin.

[0074] 4. Neartinib effectively inhibited tumor progression in MCF-7RA xenograft mice. MCF-7 parental cells and MCF-7RA resistant cells were suspended in 50% FBS (diluted with PBS) and mixed with Matrigel at a 1:1 volume ratio. After adding 0.04% trypan blue, the mixture was stored on ice. Balb / c nude mice were anesthetized, and a Y-shaped incision was made in the abdomen. The fourth pair of mammary fat pads were bluntly dissected to expose the incision site. 10-20 μL of cell suspension (MCF-7 group vs. MCF-7RA group) was injected, and the incision was sutured (screws removed 7 days post-surgery). Tumor volume (V = length × width) was monitored. 2 ×0.52), when the tumor volume in the MCF-7RA group reaches 50-100 mm. 3 Dosing in time groups: Vehicle control group: 0.5% CMC-Na solution (daily gavage); Neratinib group: 60 mg / kg (dissolved in 0.5% CMC-Na, administered by gavage daily); Treatment lasted 21 days, with tumor volume and body weight measured every 3 days. The experimental endpoint was a tumor volume <1300 mm² in the control group. 3 (In accordance with ethical thresholds), the mice were euthanized, the tumors were removed, and the tumors were weighed.

[0075] The results are as follows Figure 9As shown, a nude mouse xenograft experiment evaluated the effect of Neratinib on the growth of MCF-7 and MCF-7RA tumors. The results showed that the MCF-7RA control group (untreated) exhibited the fastest tumor growth rate, with the largest tumor volume and weight. However, treatment with Neratinib in combination with MCF-7RA significantly inhibited tumor growth, achieving a tumor inhibition rate of 83%, and the tumor weight was also significantly reduced compared to the control group. This experiment demonstrates that Neratinib can effectively inhibit the progression of MCF-7RA xenografts in vivo.

[0076] The above specific embodiments are used to explain and illustrate the present invention, and are only preferred embodiments of the present invention, not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. Use of a HER2 inhibitor in the preparation of a medicament for the treatment of CDK4 / 6 inhibitor-resistant breast cancer, wherein the HER2 inhibitor is selected from at least one or a combination of neratinib, afatinib, gefitinib, lapatinib, tucatinib, T-DM1 or trastuzumab.

2. The use according to claim 1, characterized in that: The HER2 inhibitor mentioned therein is selected from at least one of neratinib, afatinib, or gefitinib, or a combination thereof.

3. The use according to claim 2, characterized in that: The HER2 inhibitor mentioned is neratinib.

4. The use according to claim 1, characterized in that: The CDK4 / 6 inhibitor drug is selected from at least one or a combination of abecilibi, pebocilib, ribociclib, dalcilibi, or trilacimib.

5. The use according to claim 4, characterized in that: The CDK4 / 6 inhibitor drug mentioned therein is selected from abecilib, pebocillib, or ribociclib.

6. The use according to any one of claims 1-5, characterized in that: The breast cancer mentioned therein is estrogen receptor-positive breast cancer, and the breast cancer exhibits upregulated HER2 expression after developing resistance to CDK4 / 6 inhibitors.

7. A pharmaceutical composition for reversing or preventing resistance to CDK4 / 6 inhibitors in breast cancer patients, wherein the pharmaceutical composition comprises a therapeutically effective amount of a HER2 inhibitor and a CDK4 / 6 inhibitor, wherein the HER2 inhibitor is selected from at least one of neratinib, afatinib, gefitinib, lapatinib, tucatinib, T-DM1, or trastuzumab, and wherein the CDK4 / 6 inhibitor is selected from at least one of abeciclib, pebocilib, ribociclib, dalcilib, or trelacidlib.

8. The pharmaceutical composition according to claim 7, characterized in that: The HER2 inhibitor is selected from neratinib, afatinib, or gefitinib, and the CDK4 / 6 inhibitor is selected from abecicilline, pebocilline, or ribociclib.

9. The pharmaceutical composition according to claim 7, characterized in that: The HER2 inhibitor mentioned therein is neratinib, afatinib, or gefitinib, and the CDK4 / 6 inhibitor mentioned therein is abecib.

10. The pharmaceutical composition according to any one of claims 7-9, characterized in that: The breast cancer mentioned above is estrogen receptor-positive breast cancer.

11. A kit for reversing or preventing resistance to CDK4 / 6 inhibitors in breast cancer patients, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from at least one of neratinib, afatinib, gefitinib, lapatinib, tucatinib, T-DM1, or trastuzumab; and (2) Individually packaged CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitor is selected from at least one of abecicilline, pebocilline, ribocicline, dalcicilline or trilaccilline.

12. The medicine box according to claim 11, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from neratinib, afatinib or gefitinib; and (2) Individually packaged CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitors are selected from abecicilline, pebocilline or ribociclib.

13. The medicine box according to claim 12, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from neratinib, afatinib or gefitinib; and (2) Individually packaged CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitor is abecilibi.

14. The medicine box according to any one of claims 11-13, characterized in that: The breast cancer mentioned above is estrogen receptor-positive breast cancer.

15. A kit for reversing or preventing resistance to CDK4 / 6 inhibitors in patients with estrogen receptor-positive and HER2-negative breast cancer, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from at least one of neratinib, afatinib, gefitinib, lapatinib, tucatinib, T-DM1, or trastuzumab; and (2) Individually packaged CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitor is selected from at least one of abecicilline, pebocilline, ribociclib, dalcilib, or trilaccilline; The method of using the kit is as follows: first, administer a CDK4 / 6 inhibitor to the patient, and after the patient develops resistance, treat with a HER2 inhibitor to restore the sensitivity of the drug-resistant cells to the CDK4 / 6 inhibitor.

16. A kit for reversing or preventing resistance to CDK4 / 6 inhibitors in patients with estrogen receptor-positive and HER2-negative breast cancer, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from neratinib, afatinib or gefitinib; and (2) Individually packaged CDK4 / 6 inhibitors, wherein the CDK4 / 6 inhibitor is selected from abecicilline, pebocilline or ribociclib; The method of using the kit is as follows: first, administer a CDK4 / 6 inhibitor to the patient, and after the patient develops resistance, treat with a HER2 inhibitor to restore the sensitivity of the drug-resistant cells to the CDK4 / 6 inhibitor.

17. A kit for reversing or preventing resistance to CDK4 / 6 inhibitors in patients with estrogen receptor-positive and HER2-negative breast cancer, comprising: (1) Individually packaged HER2 inhibitors, wherein the HER2 inhibitor is selected from neratinib, afatinib or gefitinib; and (2) Individually packaged CDK4 / 6 inhibitor abexib; The method of using the kit is as follows: first, administer the CDK4 / 6 inhibitor abexib to the patient, and after the patient develops resistance, treat the patient with the HER2 inhibitor to restore the sensitivity of the resistant cells to the CDK4 / 6 inhibitor.

18. The medicine box according to any one of claims 15-17, characterized in that: The method of use also includes administering a CDK4 / 6 inhibitor to the patient after treatment with a HER2 inhibitor.

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