Use of sptlc1 target inhibitors in the preparation of drugs for treating lung cancer

Abstract

This invention discloses the application of an SPTLC1 target inhibitor in the preparation of drugs for treating lung cancer, which can reverse osimertinib resistance, achieve sensitization therapy, and prolong the effective time of the drug. The SPTLC1 target inhibitor enhances the sensitivity of lung cancer cells to drugs for treating lung cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of SPTLC1 target inhibitors in the preparation of drugs for treating lung cancer. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related deaths worldwide, posing a serious threat to human health. Non-small cell lung cancer (NSCLC) accounts for the vast majority of cases, and epidermal growth factor receptor (EGFR) gene mutations are a significant driving factor in lung adenocarcinoma. Osimertinib, as a third-generation EGFR-tyrosine kinase inhibitor, has become the first-line standard treatment for EGFR-mutant advanced NSCLC due to its potent inhibitory ability against classic mutations and T790M resistance mutations, significantly improving patient survival. However, acquired resistance inevitably develops with treatment, becoming a major factor limiting the clinical efficacy of osimertinib and contributing to disease progression. The resistance mechanisms of osimertinib are complex and diverse, mainly categorized into EGFR-dependent (e.g., EGFR C797S mutation) and EGFR-independent (e.g., persistent activation of key downstream signaling pathways of EGFR, histological subtype transformation, etc.). These intricate resistance mechanisms ultimately leave patients with no other treatment options after osimertinib treatment. Therefore, a thorough analysis of the resistance mechanism of osimertinib, and the search for novel biomarkers and therapeutic targets that can predict or reverse resistance, are key scientific issues that urgently need to be addressed in the field of precision treatment for lung cancer.

[0003] Sphingolipids are a diverse class of bioactive lipids with complex functions, widely involved in key biological processes such as cell membrane formation, signal transduction, and cell fate determination. Serine palmitoyltransferase (SPT) is a key enzyme in the first and rate-limiting step of de novo sphingolipid synthesis. It catalyzes the condensation of serine with palmitoyl-CoA to generate 3-ketosphingosine, a core precursor in sphingolipid synthesis. The SPT complex consists of SPTLC1 and SPTLC2 / 3; SPTLC2 / 3 contains the catalytic site, while SPTLC1 is essential for complex assembly and overall activity. For a long time, SPTLC1 was considered merely a metabolic regulator in de novo sphingolipid synthesis; however, increasing evidence suggests that SPTLC1 is dysregulated in various tumors and is closely related to tumor cell survival, proliferation, and migration. For example, SPTLC1 can inhibit the growth of renal cell carcinoma by activating the Akt / FOXO1 pathway and predict a favorable prognosis; however, it is also considered a pro-oncogene in melanoma and glioblastoma, and inhibiting SPTLC1 can suppress the proliferation of mouse B16F10 cells and human U87MG cells. Furthermore, SPTLC1 has been found to be a promoting factor for KRAS-driven tumor immune escape. However, the specific role and function of SPTLC1 in osimertinib resistance in EGFR-mutant lung adenocarcinoma remains unknown, and there are no publicly available reports, either domestically or internationally, on whether it can serve as a biomarker or intervention target for predicting or overcoming osimertinib resistance. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide an application of SPTLC1 target inhibitor in the preparation of drugs for treating lung cancer, which can reverse osimertinib resistance, achieve sensitization therapy, and prolong the effective time of the drug.

[0005] The technical solution of the present invention is: the application of an SPTLC1 target inhibitor in the preparation of drugs for treating lung cancer, wherein the SPTLC1 target inhibitor improves the sensitivity of lung cancer cells to drugs for treating lung cancer.

[0006] Preferably, the SPTLC1 target inhibitor is an SPTLC1-shRNA that inhibits the expression of the SPTLC1 gene / protein, with the sequence shown in SEQ NO 1. Specifically, the sequence is: GAUCUGAUCUUACAGUCAATT.

[0007] Preferably, the lung cancer cells are EGFR-L858R lung cancer cells.

[0008] Preferably, the lung cancer treatment drug is osimertinib.

[0009] Preferably, the SPTLC1 target inhibitor is olanzapine.

[0010] Preferably, the lung cancer is an EGFR-mutant adenocarcinoma of the lung.

[0011] Preferably, the SPTLC1 target inhibitor is used to inhibit EGFR expression in osimertinib-resistant lung cancer.

[0012] Preferably, the SPTLC1 target inhibitor is a small molecule compound inhibitor, an antibody inhibitor, or a gene editing reagent.

[0013] Preferably, the gene editing reagent is a CRISPR reagent.

[0014] Preferably, the SPTLC1 target is a molecular marker for determining osimertinib resistance in patients with EGFR-mutant lung adenocarcinoma.

[0015] This invention can significantly improve the sensitivity of drug-resistant lung cancer cells to drugs for treating lung cancer, inhibit the proliferation of drug-resistant lung cancer cells and the growth of lung cancer tumors, achieve sensitization therapy, and prolong the effective time of drugs. Attached Figure Description

[0016] Figure 1 This study demonstrates the differential protein expression of SPTLC1 in tumor tissues and adjacent normal tissues of lung cancer patients, analyzed using immunohistochemical staining, and the relationship between SPTLC1 expression and patient survival. Figure 1 A shows that SPTLC1 expression is higher in lung cancer tissue compared to adjacent normal tissue; Figure 1 B shows that higher SPTLC1 expression in lung cancer tissue is associated with poor prognosis in lung cancer patients.

[0017] Figure 2 The expression levels of SPTLC1 in osimertinib-sensitive and drug-resistant human lung cancer cells were shown based on transcriptomic data analysis of the GEO dataset.

[0018] Figure 3 The results show the protein expression levels of SPTLC1 in osimertinib-sensitive and drug-resistant lung cancer cells as detected by Western blotting. Figure 3 A shows that, compared to PC-9, osimertinib-resistant cells PC-9... del19 / T790M / C797S SPTC1 protein expression was significantly increased in the middle. Figure 3 B showed that compared with NCI-H1975, the expression of SPTLC1 protein was significantly increased in osimertinib-resistant cells NCI-H1975 / OR.

[0019] Figure 4 The study demonstrated the effects of osimertinib treatment on the growth of drug-resistant tumors and SPTLC1 expression. Figure 4 A shows the tumor growth curves for each group; Figure 4 B shows the expression of SPTC1 and EGFR proteins in the two groups of tumor tissues as detected by immunohistochemical staining.

[0020] Figure 5 The effect of SPTLC1 on the sensitivity of lung cancer cells to osimertinib was demonstrated. Figure 5 A shows the construction of a stable SPTLC1 knockdown PC-9 cell line and its control cells using lentivirus. Cell viability after treatment with different concentrations of osimertinib was detected using the CCK8 assay, and the IC50 was calculated. 50 value. Figure 5 B shows the use of lentivirus to construct osimertinib-resistant PC-9 cells with stable SPTLC1 knockdown. del19 / T790M / C797S The IC50 of osimertinib-treated cells and control cells were analyzed using the CCK8 assay. 50 value.

[0021] Figure 6 This study demonstrates the effect of SPTLC1 knockdown and osimertinib treatment on PC-9 using a plate colony formation assay. del19 / T790M / C797S Effects on cell proliferation capacity.

[0022] Figure 7 The interaction between SPTLC1 and EGFR and the effect of the SPTLC1 inhibitor olanzapine are shown.

[0023] Figure 8 The study demonstrated the effects of SPTLC1 inhibitor combined with osimertinib on the growth of NCI-H1975 lung cancer subcutaneous xenografts and EGFR expression. Figure 8 A shows the tumor growth curves for each group; Figure 8 B shows the expression levels of SPTLC1 and EGFR in tumor tissues of each group, as detected by immunohistochemical staining. Detailed Implementation

[0024] This invention creatively discovers that SPTLC1, a key enzyme in sphingolipid synthesis, can serve as a molecular marker for assessing osimertinib resistance in EGFR-mutant lung adenocarcinoma patients, enabling the screening and development of personalized treatment regimens to reverse resistance. This invention finds that SPTLC1 expression is higher in osimertinib-resistant lung cancer cells compared to osimertinib-sensitive cells. Inhibiting SPTLC1 expression significantly enhances the sensitivity of osimertinib-resistant lung cancer cells, inhibits the proliferation of resistant lung cancer cells and the growth of lung cancer tumors, and downregulates EGFR expression levels. Therefore, substances that inhibit SPTLC1 gene expression and / or function in lung cancer cells can reverse osimertinib resistance, providing a new therapeutic target for the treatment of osimertinib-resistant lung cancer. This target can be used to prepare combination drugs that enhance osimertinib therapy, thereby improving the treatment efficacy and prognosis of EGFR-mutant lung cancer patients.

[0025] The embodiments of the present invention will be described in detail below.

[0026] Experimental materials:

[0027] 1. Cell lines: PC-9 cells and NCI-H1975 cells were purchased from Wuhan Pronosei Life Sciences Co., Ltd.; PC-9 del19 / T790M / C797S Purchased from Nanjing Kebai Biotechnology Co., Ltd.; NCI-H1975 / OR cells purchased from Zhejiang Meisen Cell.

[0028] 2. Experimental reagents and consumables: CCK-8 reagent kit (catalog number: CK04) was purchased from DOJINDO; protein extraction and lysis buffer RIPA (catalog number: P0013B) was purchased from Beyotime Biotechnology Co., Ltd.; protein inhibitor cocktail (PIC) (catalog number: P1005) was purchased from Beyotime Biotechnology Co., Ltd.; PMSF (catalog number: A1060) was purchased from Applygen. BCA (catalog number: PK10026) was purchased from Proteintech Co., Ltd.; human lung cancer tumor tissue microarray (HLugA180Su07) was purchased from Shanghai Chipchao Biotechnology Co., Ltd.; osimertinib and olanzapine were purchased from MCE. Example 1: Study on the relationship between SPTLC1 protein expression and patient prognosis using human lung cancer tumor tissue microarray.

[0029] Immunohistochemical staining of tumor tissue microarray (HLugA180Su07) from human lung cancer patients was used to analyze the differential expression of SPTLC1 protein in tumor tissue and adjacent normal tissue of lung cancer patients, and the relationship between SPTLC1 protein expression and survival rate of lung cancer patients.

[0030] The results showed that SPTLC1 expression was higher in lung cancer tissue compared to adjacent normal tissue. Figure 1 A); and higher SPTLC1 expression in lung cancer tissue is associated with poor prognosis in lung cancer patients (A); Figure 1 B). Example 2: SPTLC1 Expression Analysis Based on GEO Dataset

[0031] Transcriptomic data analysis was performed on human lung cancer osimertinib-sensitive cells NCI-H1975 and human lung cancer osimertinib-resistant cells NCI-H1975 / Osimertinib resistance (H1975 / OR) based on the GEO dataset (GSE193258). The results showed that SPTLC1 expression was significantly increased in both osimertinib-sensitive and osimertinib-resistant cells compared to lung cancer-sensitive cells. Figure 2 ). Example 3: SPTLC1 protein expression in osimertinib-sensitive and osimertinib-resistant lung cancer cells

[0032] Lung cancer sensitive cells (PC-9 (EGFR del19) and NCI-H1975 (EGFR L858R-T790M)) and corresponding osimertinib-resistant lung cancer cells (PC-9) were collected in good growth condition and in the logarithmic growth phase. del19 / T790M / C797S (Compared with NCI-H1975 / OR), digested with 0.25% trypsin and diffused into single cells. Following a 1*10 6 Cells were seeded at a density of [number] cells / well in six-well plates. After 24 hours of cell adhesion, cells were collected, washed twice with pre-chilled PBS, and transferred to 1.5 ml centrifuge tubes. 200 μl of protein extraction lysis buffer (RIPA lysis buffer with added PIC and PMSF) was added. Lysis was performed on ice for 30 min, followed by centrifugation at 12000 rpm for 30 min at 4°C. The supernatant was collected, and protein was quantified using the BCA method, adjusting the protein concentration to the same level. 5×SDS gel loading buffer was added, mixed, and denatured at 98°C for 10 min. Western blotting was then performed to detect SPTLC1 protein expression in lung cancer-sensitive and drug-resistant cells.

[0033] The results showed that, compared with osimertinib-sensitive PC-9 lung cancer cells, drug-resistant PC-9 cells... del19 / T790M / C797S SPTLC1 protein expression was significantly increased in the middle ( Figure 3 A). Similarly, compared with osimertinib-sensitive lung cancer cells NCI-H1975, SPTLC1 protein expression was significantly increased in drug-resistant cells NCI-H1975 / OR (A). Figure 3 B). Example 4: Effects of osimertinib treatment on the growth of drug-resistant tumors and SPTLC1 expression

[0034] PC-9 patients resistant to osimertinib del19 / T790M / C797S A subcutaneous xenograft tumor model of NCG mice was constructed, and PC-9 was used. del19 / T790M / C797S Cells were inoculated into the axillae of NCG mice, and tumor growth was observed daily until the tumor volume reached 80-100 mm. 3 Mice were then divided into groups for drug administration. The osimertinib group received osimertinib once daily by gavage (10 mg / kg), while the control group received saline. After 25 days, mice were sacrificed by cervical dislocation, tumors were removed, tumor growth curves were plotted, and SPTLC1 expression was detected by immunohistochemical staining.

[0035] The results showed that, compared with the saline group, the osimertinib group inhibited the growth of drug-resistant tumors. Figure 4 A), and SPTLC1 and EGFR expression were significantly increased in the osimertinib group ( Figure 4 B).

[0036] The above experiments suggest that under the pressure of osimertinib treatment, the expression of SPTLC1 and EGFR in drug-resistant tumors is simultaneously upregulated, and the two may synergistically participate in the maintenance of acquired resistance to osimertinib. Example 5: Knocking down SPTLC1 in lung cancer cells to improve the sensitivity of lung cancer cells to osimertinib

[0037] 1. A PC-9 cell line with stable SPTLC1 knockdown (shRNA sequence: GAUCUGAUCUUACAGUCAATT) and its control cells were constructed using lentivirus. The IC50 of osimertinib-treated osimertinib-resistant PC-9 cells and stably knocked-down SPTLC1 PC-9 cells was detected using CCK8 assay. 50 Add 10% CCK-8 solution to each well, place the culture plate in an incubator and continue incubation for 1-4 hours, measure the absorbance at 450 nm using an ELISA reader, and calculate the cell viability (%) = (absorbance of experimental group - absorbance of blank group) / (absorbance of control group - absorbance of blank group) * 100%.

[0038] The results showed that after SPTLC1 knockdown, the IC50 of osimertinib-treated PC-9 cells was reduced. 50 The value decreased significantly by approximately 2.34 times. Figure 5 A) indicates that PC-9 cells are more sensitive to osimertinib after SPTLC1 knockdown.

[0039] 2. Construct a stable PC-9 system to knock down SPTLC1 using lentivirus. del19 / T790M / C797S Cell lines and control cells. CCK8 assay was used to detect osimertinib-treated osimertinib-resistant lung cancer cells PC-9. del19 / T790M / C797S and PC-9 for stable knockdown of SPTLC1 del19 / T790M / C797S Cellular IC 50 Add 10% CCK-8 solution to each well, place the culture plate in an incubator and continue incubation for 1-4 hours, measure the absorbance at 450 nm using an ELISA reader, and calculate the cell viability.

[0040] The results showed that after SPTLC1 knockdown, the IC50 of osimertinib-treated drug-resistant lung cancer cells was significantly reduced. 50 The value decreased significantly by approximately 4.39 times. Figure 5 B) indicates that knocking down SPTLC1 can significantly enhance the performance of drug-resistant lung cancer cells PC-9. del19 / T790M / C797S Sensitivity to osimertinib suggests that SPTLC1 plays an important role in acquired resistance to osimertinib. Example 6: Knocking down SPTLC1 to inhibit the clonogenic ability of drug-resistant lung cancer cells

[0041] Take lung cancer drug-resistant PC-9 that is in good growth condition and in the logarithmic growth phase. del19 / T790M / C797S Cells and PC-9 cells with stable SPTLC1 knockdown del19 / T790M / C797S Cell lines were digested with 0.25% trypsin and dispersed into single cells. Cells were seeded at a density of 500 cells / well in six-well plates. The cells were divided into two groups: a control group and a group treated with osimertinib.

[0042] The results showed that after knocking down the SPTLC1 gene, PC-9 del19 / T790M / C797S Cell colony-forming ability was significantly reduced; and compared with the osimertinib treatment group, the colony-forming ability of cells knocked down SPTLC1 and treated with osimertinib was further reduced. Figure 6 ). Example 7: Olanzapine, a SPTLC1 inhibitor, disrupts the interaction between SPTLC1 and EGFR, thereby inhibiting EGFR expression.

[0043] Two plates of PC-9 cells in good growth and logarithmic growth phase, and one plate of PC-9 cells treated with the SPTLC1 inhibitor olanzapine were collected. All were incubated on ice for 30 min with CO-IP lysis buffer. Centrifuged at 12000 rpm for 30 min. 60 μl of protein sample was added to 15 μl of 5× loading buffer and denatured at 98°C for 10 min. The remaining protein sample was used as an IP treatment, incubated overnight with IgG or EGFR antibody and protein A / G beads. The cells were washed five times with COIP wash buffer, incubated at 98°C for 10 min with 20 μl of 2× loading buffer, and then subjected to Western blotting.

[0044] CO-IP results showed an interaction between SPTLC1 and EGFR proteins; treatment with the SPTLC1 inhibitor olanzapine significantly weakened this interaction. Simultaneously, input results showed that olanzapine reduced EGFR protein expression levels. Figure 7 ). Example 8: SPTLC1 inhibitors enhance the sensitivity of lung cancer tumors to osimertinib

[0045] A subcutaneous xenograft tumor model in NCG mice was constructed using osimertinib-sensitive NCI-H1975 (EGFR L858R / T790M). NCI-H1975 cells were inoculated into the axillae of NCG mice, and tumor growth was observed daily until the tumor volume reached 80-100 mm². 3Mice were then divided into four groups: a control group, an osimertinib group (5 mg / kg orally once daily), an olanzapine group (0.75 mg / kg orally once daily), and an olanzapine group combined with osimertinib. After 25 days, mice were sacrificed by cervical dislocation, tumors were removed, tumor growth curves were plotted, and immunohistochemical staining was used to detect the expression of SPTLC1 and EGFR in the tumor tissues of each group.

[0046] The results showed that, compared with the osimertinib treatment group, the combination of SPTLC1 inhibitor and osimertinib significantly reduced the growth of lung cancer tumors. Figure 8 A). Simultaneously, immunohistochemical staining results showed that EGFR expression decreased after using the SPTLC1 inhibitor, and EGFR expression further decreased after combining it with osimertinib. Figure 8 B). The above data suggest that SPTLC1 inhibitors may increase the sensitivity of lung cancer tumors to osimertinib by inhibiting EGFR expression.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. The application of SPTLC1 target inhibitors in the preparation of drugs for treating lung cancer, characterized in that: SPTLC1 target inhibitors improve the sensitivity of lung cancer cells to drugs for treating lung cancer.

2. The application of the SPTLC1 target inhibitor according to claim 1 in the preparation of drugs for treating lung cancer, characterized in that: The SPTLC1 target inhibitor is an SPTLC1-shRNA that inhibits the expression of the SPTLC1 gene / protein, and its sequence is shown in SEQ NO1.

3. The application of the SPTLC1 target inhibitor according to claim 2 in the preparation of drugs for treating lung cancer, characterized in that: The lung cancer cells were EGFR-L858R lung cancer cells.

4. The application of the SPTLC1 target inhibitor according to claim 1 in the preparation of drugs for treating lung cancer, characterized in that: The drug used to treat lung cancer is osimertinib.

5. The application of the SPTLC1 target inhibitor according to claim 1 in the preparation of drugs for treating lung cancer, characterized in that: The lung cancer in question is an EGFR-mutant adenocarcinoma of the lung.

6. The application of the SPTLC1 target inhibitor according to claim 5 in the preparation of drugs for treating lung cancer, characterized in that: The SPTC1 target inhibitor is used to inhibit EGFR expression in osimertinib-resistant lung cancer.

7. The application of the SPTLC1 target inhibitor according to claim 1 in the preparation of drugs for treating lung cancer, characterized in that: The SPTLC1 target inhibitor is a small molecule compound inhibitor, an antibody inhibitor, or a gene editing reagent.

8. The application of the SPTLC1 target inhibitor according to claim 7 in the preparation of a drug for treating lung cancer, characterized in that: The gene editing reagent is a CRISPR reagent.

9. The application of the SPTLC1 target inhibitor according to claim 1 in the preparation of drugs for treating lung cancer, characterized in that: The SPTC1 target is a molecular marker for determining osimertinib resistance in patients with EGFR-mutant lung adenocarcinoma.

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