Targeted YY1 S247 phosphorylated shuttle peptide and application thereof

Abstract

The invention discloses a shuttle peptide targeting YY1S247 phosphorylation and an application of the shuttle peptide. The sequence of the shuttle peptide is as shown in SEQ ID NO: 1. Wherein the RQIKIWFQNRRMKWKK is a part of the drosophila melanogaster contact foot peptide and is used for providing cell penetrability; the PKKKRKV is a nuclear localization sequence of the SV40 large T antigen and is used for guiding a peptide fragment to enter a cell nucleus; and the QIIGENSPPDYSE is a peripheral sequence of the YY1S247 site. Results show that the shuttling peptide (CPP) can competitively, specifically and effectively inhibit phosphorylation of the YY1S247 site, so that CD24 expression is inhibited, and the treatment effect of EGFR-TKI is enhanced.

Description

Technical Field

[0001] The invention relates to a shuttle peptide targeting YY1 S247 phosphorylation and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] EGFR (epidermal growth factor receptor) mutation is an important driver gene for non-small cell lung cancer. EGFR-TKI (tyrosine kinase inhibitor) is currently the main treatment for EGFR mutation non-small cell lung cancer. It binds to the mutated EGFR and blocks its cancer-promoting effect. At present, the third-generation EGFR-TKI represented by osimertinib has become the first-line drug for the treatment of EGFR-positive lung cancer worldwide, significantly improving patient survival. Generally, in the initial stage of treatment, the third-generation EGFR-TKI has an excellent effect, but there will always be a small number of residual tumor cells that cannot be cured, and these residual cells will eventually develop drug resistance, resulting in tumor progression after a median time of about 10-20 months. Although immunotherapy has achieved good results in a variety of tumors, the most commonly used immune checkpoint inhibitors in lung cancer clinics are currently ineffective when used alone or in combination with EGFR-TKI to treat patients with EGFR mutation lung cancer. In order to further enhance the efficacy and improve the prognosis of lung cancer patients, the development of a new immunotherapy regimen combined with the third-generation EGFR-TKI treatment is still a key scientific issue facing current medicine.

[0003] CD24 is a cell surface antigen that is highly expressed in a variety of tumor cells, including NSCLC. Studies have shown that high expression of CD24 is associated with drug resistance and poor prognosis in patients with EGFR-mutated NSCLC. CD24 promotes the growth, proliferation, metastasis and invasion of tumor cells by participating in related signal transduction pathways that mediate tumor occurrence and development, such as the Wnt signaling pathway and the MAPK signaling pathway. In addition, CD24 can also escape immune surveillance by binding to Siglec-10 on macrophages and releasing signals that inhibit macrophages from phagocytosing tumor cells.

[0004] YY1 (Yin Yang 1) is a multifunctional transcription factor widely present in eukaryotes. It regulates the expression of multiple genes and is closely related to cell proliferation, differentiation, apoptosis, and tumor resistance. The activity of YY1 is regulated by a variety of post-translational modifications, including phosphorylation and acetylation. The phosphorylation of the serine residue at position 247 (S247) of the YY1 protein has rarely been reported, and its function has not been fully revealed. In particular, there have been no reports on the regulation of CD24 expression by YY1S247 phosphorylation, the sensitization effect of EGFR-TKI, and targeted intervention measures for YY1 S247 phosphorylation. Summary of the invention

[0005] The purpose of the present invention is: in view of the deficiencies of the prior art, the present invention provides a shuttle peptide targeting YY1 S247 phosphorylation, and the shuttle peptide can competitively inhibit YY1 S247 phosphorylation.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a shuttle peptide targeting YY1 S247 phosphorylation, wherein the amino acid sequence of the shuttle peptide is shown in SEQ ID NO:1.

[0008] In a second aspect, the present invention provides use of the aforementioned shuttle peptide in the preparation of a product for inhibiting phosphorylation of YY1 S247 site.

[0009] In a third aspect, the present invention provides use of the aforementioned shuttle peptide in the preparation of a product for inhibiting CD24 expression.

[0010] In a fourth aspect, the present invention provides the use of the aforementioned shuttle peptide in the preparation of a drug for enhancing the efficacy of EGFR-TKI (tyrosine kinase inhibitor).

[0011] In a fifth aspect, the present invention provides use of the aforementioned shuttle peptide in the preparation of a drug for treating lung adenocarcinoma with EGFR mutation.

[0012] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the shuttle peptide as described above and EGFR-TKI (tyrosine kinase inhibitor).

[0013] Preferably, the EGFR-TKI is a third-generation EGFR-TKI, and the third-generation EGFR-TKI is selected from at least one of osimertinib, ametinib, vometinib, befortinib, rizitinib and rieutinib.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The shuttle peptide provided by the present invention can effectively penetrate the cell membrane and the nuclear membrane without adding a transfection reagent, enter the cell nucleus, and competitively and specifically inhibit the phosphorylation of the YY1 S247 site, thereby inhibiting CD24 expression and enhancing the therapeutic effect of EGFR-TKI; the uses of the shuttle peptide provided by the present invention include but are not limited to reducing CD24 expression and enhancing the therapeutic effect of EGFR-TKI (enhancing the efficacy of the third-generation EGFR-TKI on EGFR-mutated lung adenocarcinoma). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The results of qRT-PCR detection of three cell lines PC-9, HCC827 and H1975 (A) and drug-resistant persistent (DTP) cells (B) after short-term treatment with the third-generation EGFR-TKI, and Western blot detection of CD24 expression in short-term treated drug-resistant cells (C) were shown. The results showed that CD24 expression was significantly increased.

[0017] Figure 2 The results show that CD24 knockout promotes the resistance of EGFR-mutated non-small cell lung cancer cells PC-9 (A), HCC827 (B), and H1975 (C) to four third-generation EGFR-TKIs.

[0018] Figure 3 The results showed that CD24 knockout significantly promoted the phagocytosis of non-small cell lung cancer cells PC-9 (A) and H1975 (B) after treatment with the third-generation EGFR-TKI by macrophages.

[0019] Figure 4 The figure shows that YY1 regulates CD24 expression; A: transcription factors screened by DNA affinity precipitation / mass spectrometry experiment of CD24 promoter probe; B: YY1 binding peak on CD24 promoter region in ENCODE database; CE: ChIP-qPCR (C), affinity precipitation / Western blot (D), and dual luciferase (E) experimental results of YY1 on CD24 promoter region.

[0020] Figure 5 The expression of YY1 after third-generation EGFR-TKI treatment is shown; A: qRT-PCR results; B: Western blot results.

[0021] Figure 6 The third-generation EGFR-TKI promotes CD24 expression by inducing phosphorylation of YY1 S247; A: Mass spectra of YY1 S247 phosphorylation in PC-9 parental and resistant cells; B: Western blot detection results of YY1 S247 phosphorylation level after treatment with third-generation EGFR-TKI; C: Changes in YY1 S247 phosphorylation level in parental and osimertinib-resistant cells; D-E: qRT-PCR (D) and Western blot (E) results showing that YY1 S247A mutation blocks the promotion of CD24 expression by third-generation EGFR-TKI.

[0022] Figure 7The effects of shuttle peptides targeting YY1 S247 are shown; A: The inhibitory effect of shuttle peptides on YY1 S247 phosphorylation and CD24 expression; B: The sensitizing effect of shuttle peptides on the third-generation EGFR-TKI; C: Shuttle peptides promote macrophages to phagocytose tumor cells treated with the third-generation EGFR-TKI. DETAILED DESCRIPTION

[0023] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0024] Example 1

[0025] In this example, four third-generation EGFR-TKIs (osimertinib, ametinib, vometinib, befortinib) were used to treat EGFR mutation non-small cell lung cancer cell lines (PC-9, HCC827, H1975) for 48 hours, or to treat them for 10 days to construct drug-resistant persistent (DTP) cells, or to treat them for 3 months with osimertinib to construct drug-resistant cells, and qRT-PCR and Western blot were used for detection. It was found that the third-generation EGFR-TKI treatment led to a significant increase in CD24 expression in both short-term treatment, drug-resistant persistent cells, and drug-resistant cells, as shown in Figure 2. Figure 1 As shown in AC.

[0026] Furthermore, to explore the effect of CD24 expression on the efficacy of EGFR-TKI, drug resistance studies were conducted in PC-9, HCC827 and H1975 cell lines with CD24 gene knockout (ΔCD24). The results showed that compared with wild-type cells, ΔCD24 cells were significantly more sensitive to third-generation EGFR-TKIs, such as Figure 2 As shown in AC.

[0027] Given that CD24 is an important signaling molecule against macrophage phagocytosis "don't eat me", we sought to explore whether upregulation of CD24 caused by third-generation EGFR-TKI treatment induced tumor immune escape. To measure phagocytic clearance, we labeled lung cancer cells with CFSE and co-cultured them with THP-1-derived macrophages. The results showed that knocking out CD24 significantly promoted the phagocytosis of non-small cell lung cancer cells treated with third-generation EGFR-TKI by macrophages, such as Figure 3 As shown in AB.

[0028] Given that CD24 was significantly increased at both mRNA and protein levels under treatment with third-generation EGFR-TKIs, we hypothesized that transcription factors might be responsible for the increased CD24 expression after treatment. Therefore, we combined DNA affinity precipitation experiments with mass spectrometry (MS) to search for TFs that might regulate CD24 in PC-9 cells and identified 23 candidate factors ( Figure 4 A). Based on ChIP sequencing data in the ENCODE database, we found that the YY1 transcription factor (YY1) has a significant binding peak in the CD24 promoter region ( Figure 4 B). Our ChIP-qPCR, DNA affinity precipitation / Western blot and dual luciferase assays all showed that YY1 significantly regulated CD24 expression ( Figure 4 CE).

[0029] However, the third-generation EGFR-TKI treatment did not significantly change the expression of YY1 ( Figure 5 AB).

[0030] Example 2

[0031] Considering that post-translational modification of proteins also plays a key role in protein function, we performed YY1 CoIP combined with mass spectrometry experiments in PC-9 parental and drug-resistant cells. The analysis results showed that the S247 phosphorylation of YY1 was significantly increased in drug-resistant cells ( Figure 6 A). Further using a custom antibody targeting YY1 S247 phosphorylation and an antibody specific for the unique phosphorylated Ser-Pro-Pro motif surrounding the S247 residue, we determined that the third-generation EGFR-TKI induced a significant increase in the phosphorylation of YY1 S247 site ( Figure 6 BC).

[0032] To examine whether YY1 S247 phosphorylation is a key factor in the increase in CD24 expression induced by the third-generation EGFR-TKI, we used a cell line with the YY1 S247A mutation knocked in. The results showed that the S247A mutation significantly reduced the expression of CD24 and blocked the increase in CD24 expression caused by the third-generation EGFR-TKI ( Figure 6 DE). These results suggest that EGFR-TKI promotes CD24 expression by inducing phosphorylation of YY1S247.

[0033] Therefore, we considered using a shuttle peptide targeting YY1 S247 phosphorylation to specifically and competitively inhibit this phosphorylation, thereby inhibiting CD24 expression, enhancing the effect of the third-generation EGFR-TKI and the phagocytosis of macrophages on cells treated with the third-generation EGFR-TKI. The corresponding sequence is RQIKIWFQNRRMKWKKPKKKRKVQIIGENSPPDYSE (SEQID NO: 1). Among them, RQIKIWFQNRRMKWKK (SEQ ID NO: 2) is a part of the Drosophila melanogaster antenna foot peptide, which is used to provide cell permeability. PKKKRKV (SEQ ID NO: 3) is the nuclear localization sequence of the SV40 large T antigen, which is used to guide the peptide segment into the cell nucleus. QIIGENSPPDYSE (SEQ ID NO: 4) is the surrounding sequence of the YY1 S247 site. The results showed that the shuttle peptide (CPP) of the present invention can competitively and specifically inhibit the phosphorylation of YY1 S247 site, thereby inhibiting CD24 expression and enhancing the therapeutic effect of EGFR-TKI ( Figure 7 AC).

[0034] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A shuttle peptide targeting YY1 S247 phosphorylation, characterized in that: The amino acid sequence of the shuttle peptide is shown in SEQ ID NO:

1.

2. Use of the shuttle peptide according to claim 1 in the preparation of a product for inhibiting phosphorylation of YY1 S247 site.

3. Use of the shuttle peptide according to claim 1 in the preparation of a product for inhibiting CD24 expression.

4. Use of the shuttle peptide according to claim 1 in the preparation of a drug for enhancing the efficacy of EGFR-TKI (tyrosine kinase inhibitor).

5. Use of the shuttle peptide according to claim 1 in the preparation of a drug for treating lung adenocarcinoma with EGFR mutation.

6. A pharmaceutical composition, characterized in that It comprises the shuttle peptide according to claim 1 and EGFR-TKI (tyrosine kinase inhibitor).

7. The pharmaceutical composition according to claim 6, characterized in that The EGFR-TKI is a third-generation EGFR-TKI, and the third-generation EGFR-TKI is selected from at least one of osimertinib, ametinib, vometinib, befortinib, rizitinib and rieutinib.

Images