Application of composition in preparation of medicine for treating myeloblastoma

Abstract

The invention provides application of a composition in preparation of a medicine for treating medulloblastoma, the composition comprises a substance for inhibiting FOXD1 and a substance for inhibiting PD-1, the substance for inhibiting FOXD1 can effectively enhance the effectiveness of the substance for inhibiting PD-1, and a new strategy is provided for individualized treatment of medulloblastoma.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular, to an application of a composition in preparing a drug for treating medulloblastoma and a drug for treating medulloblastoma. Background Art

[0002] Medulloblastoma (MB) is the most common malignant pediatric brain tumor in the cerebellum. Currently, medulloblastoma can be divided into four molecular subtypes: WNT subtype, SHH subtype (with or without TP53 mutation), and non-WNT / SHH subtype. About 30% of medulloblastomas show overactivated hedgehog (SHH) signaling pathway. Compared with other subtypes, SHH-MB has more TAMs infiltration in the tumor microenvironment (TME), which may be related to the formation of suppressive immune microenvironment (IME), resulting in reduced sensitivity to immunotherapy, but the mechanism is still unclear.

[0003] The current combined treatment strategy of surgical treatment and radiotherapy and chemotherapy has brought the overall 5-year survival rate of patients to more than 50%. However, since the intellectual damage caused by high-dose radiotherapy seriously affects the quality of life of children and their families, molecular targeted therapy and individualized precision treatment have become the most concerned directions in the field of medulloblastoma research. SHH-type medulloblastoma has unique molecular characteristics and may have significant differences in clinical manifestations, treatment response, etc. from other medulloblastoma subtypes.

[0004] Therefore, it is urgent to discover new molecular markers that can improve the therapeutic sensitivity of medulloblastoma and provide new strategies for personalized treatment. Summary of the invention

[0005] The present disclosure provides an application of the composition in the preparation of a drug for treating medulloblastoma. The drug can significantly improve the immunotherapy sensitivity of SHH-MB and provide a new strategy for the treatment of medulloblastoma.

[0006] In one aspect, the present disclosure provides use of a composition for preparing a medicament for treating medulloblastoma, wherein the composition comprises a substance that inhibits FOXD1 and a substance that inhibits PD-1.

[0007] Optionally, the medulloblastoma is SHH-type medulloblastoma.

[0008] Optionally, the substance that inhibits FOXD1 includes siRNA targeting FOXD1, shRNA targeting FOXD1, antisense nucleotides targeting FOXD1, antibodies against FOXD1, and small molecule compounds that inhibit FOXD1; The substance that inhibits PD-1 includes siRNA targeting PD-1, shRNA targeting PD-1, antisense nucleotides targeting PD-1, anti-PD-1 antibodies and small molecule compounds that inhibit PD-1, preferably anti-PD-1 antibodies.

[0009] Optionally, the shRNA targeting FOXD1 includes shFOXD1; the forward and reverse strands of the nucleotide sequence of the shFOXD1 are shown in SEQ ID NO.1 and SEQ ID NO.2; CCGGGCCCTTCTCCATCGAGAGCATSEQ ID NO.1, CCGGCGAGGAAACAGACATCGACGTSEQ ID NO.2.

[0010] Optionally, the dosage form of the drug includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.

[0011] In another aspect, the present disclosure provides a drug for treating medulloblastoma, the drug comprising a substance that inhibits FOXD1 and a substance that inhibits PD-1; The substance that inhibits FOXD1 includes siRNA targeting FOXD1, shRNA targeting FOXD1, antisense nucleotides targeting FOXD1, antibodies against FOXD1 and small molecule compounds that inhibit FOXD1; the substance that inhibits PD-1 includes siRNA targeting PD-1, shRNA targeting PD-1, antisense nucleotides targeting PD-1, antibodies against PD-1 and small molecule compounds that inhibit PD-1, preferably antibodies against PD-1.

[0012] Optionally, the medulloblastoma is SHH-type medulloblastoma.

[0013] Optionally, the shRNA targeting FOXD1 includes shFOXD1; the forward and reverse strands of the nucleotide sequence of the shFOXD1 are shown in SEQ ID NO.1 and SEQ ID NO.2; CCGGGCCCTTCTCCATCGAGAGCATSEQ ID NO.1, CCGGCGAGGAAACAGACATCGACGTSEQ ID NO.2.

[0014] In the drug, the content of the substance inhibiting FOXD1 is 20-50 nM, and the content of the substance inhibiting PD-1 is 10-100 nM.

[0015] The dosage form of the drug includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.

[0016] Through the above technical scheme, the present disclosure provides the use of a composition in the preparation of a drug for treating medulloblastoma, wherein the composition includes a substance that inhibits FOXD1 and a substance that inhibits PD-1, wherein the substance that inhibits FOXD1 can effectively enhance the effectiveness of the substance that inhibits PD-1, thereby achieving individualized and precise treatment of medulloblastoma.

[0017] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 The protein expression of FOXD1 in Daoy cells treated with shVector and shFOXD1-1 / 2 / 3; Figure 2 Heatmap of normalized gene expression levels for shVector- and shFOXD1-2 / 3-treated Daoy cells; Figure 3 Volcano plot of differential gene expression in Daoy cells treated with shVector and shFOXD1-3; Figure 4 Overlap determination diagram between shFOXD1-2 transcripts and shFOXD1-3 transcripts in Daoy cells; Figure 5 Metascape enrichment analysis of 2181 genes in the whole transcriptome of Daoy cells with knockdown of FOXD1; Figure 6 qRT-PCR detection graphs of the expression levels of CXCL10 / 11, IFNL1, TNFAIP3, CCL5, CCL20, ISG15, and FOXD1; Figure 7 ELISA test graphs for the expression levels of CXCL10 / 11, IFNL1, TNFAIP3, CCL5, CCL20, ISG15, and FOXD1; Figure 8qRT-PCR detection of the relative expression levels of CXCL10 / 11, IFNL1, TNFAIP3, CCL5, CCL20, ISG15 and FOXD1 in Daoy cells after treatment with THP-1-derived M0 / 2-EXO or M2-EXO+shFOXD1; Fig. 9 ELISA detection graph of the relative expression levels of CXCL10 / 11, IFNL1, TNFAIP3, CCL5, CCL20, ISG15 and FOXD1 in Daoy cells after treatment with THP-1-derived M0 / 2-EXO or M2-EXO + shFOXD1; Fig.10 Schematic diagram of in vivo experiments and single and combined administration of AAV2 / 9-shFOXD1 and PD-1 blocking antibodies; Fig.11 Representative magnetic resonance imaging (MRI) images of each animal in each group on days 0, 20, 40, and 60; Fig.12 The tumor volume of mice in different treatment groups during the administration period was measured; Fig.13 It is the Kaplan-Meier survival curve determination; Fig.14 Representative immunohistochemical staining images of CD8a, CD163, CXCL10, and CXCL11 in different treatment groups. DETAILED DESCRIPTION

[0019] The specific embodiments of the present disclosure are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.

[0020] In one aspect, the present disclosure provides use of a composition for preparing a medicament for treating medulloblastoma, wherein the composition comprises a substance that inhibits FOXD1 and a substance that inhibits PD-1.

[0021] The medulloblastoma is SHH type medulloblastoma.

[0022] Optionally, the substance that inhibits FOXD1 includes siRNA targeting FOXD1, shRNA targeting FOXD1, antisense nucleotides targeting FOXD1, antibodies against FOXD1 and small molecule compounds that inhibit FOXD1; the substance that inhibits PD-1 includes siRNA targeting PD-1, shRNA targeting PD-1, antisense nucleotides targeting PD-1, antibodies against PD-1 and small molecule compounds that inhibit PD-1, preferably antibodies against PD-1.

[0023] Optionally, the shRNA targeting FOXD1 includes shFOXD1; the forward and reverse strands of the nucleotide sequence of the shFOXD1 are shown in SEQ ID NO.1 and SEQ ID NO.2; CCGGGCCCTTCTCCATCGAGAGCATSEQ ID NO.1, CCGGCGAGGAAACAGACATCGACGTSEQ ID NO.2.

[0024] Optionally, the dosage form of the drug includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.

[0025] In another aspect, the present disclosure provides a drug for treating medulloblastoma, the drug comprising a substance that inhibits FOXD1 and a substance that inhibits PD-1.

[0026] The substance that inhibits FOXD1 includes siRNA targeting FOXD1, shRNA targeting FOXD1, antisense nucleotides targeting FOXD1, antibodies against FOXD1, and small molecule compounds that inhibit FOXD1; the substance that inhibits PD-1 includes siRNA targeting PD-1, shRNA targeting PD-1, antisense nucleotides targeting PD-1, antibodies against PD-1, and small molecule compounds that inhibit PD-1, preferably antibodies against PD-1; The medulloblastoma is SHH type medulloblastoma.

[0027] Optionally, the shRNA targeting FOXD1 includes shFOXD1; the forward and reverse strands of the nucleotide sequence of the shFOXD1 are shown in SEQ ID NO.1 and SEQ ID NO.2; CCGGGCCCTTCTCCATCGAGAGCATSEQ ID NO.1, CCGGCGAGGAAACAGACATCGACGTSEQ ID NO.2.

[0028] Optionally, in the drug, the content of the substance inhibiting FOXD1 is 20-50 nM, and the content of the substance inhibiting PD-1 is 10-100 nM.

[0029] Optionally, the dosage form of the drug includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.

[0030] The present invention is further described in detail below by way of examples.

[0031] Example 1 This example is used to illustrate the discovery of FOXD1 targets and in vitro experiments to verify that inhibiting the expression of FOXD1 can reshape the SHH-MB tumor microenvironment.

[0032] (1) Patients and samples: This publication was approved by the Institutional Review Board of Peking University (No. IRB00006761-M2023465). All 200 samples were diagnosed and histologically subtyped by two neuropathologists (QC and DZ), and molecular subtypes were classified using the NanoString nCounter Analysis System (NanoString Technologies) in the Department of Pathology, Peking University Third Hospital. Human medulloblastoma (MB) Daoy cell line (HTB-186, ATCC) and THP-1 cell line (TIB-202, ATCC) were purchased from the Cell Resource Center of Peking Union Medical College. Human ONS-76 (IFO50355, JCRB) MB cell line was provided by the Southwest Hospital of Army Medical University. Daoy, ONS-76, and THP-1 were cultured in MEM and RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. All cells were tested negative for mycoplasma contamination before use. Cells were cultured in a humidified incubator at 37°C and balanced with 5% CO . Patient and tumor characteristics are shown in Table 1 .

[0033] Table 1

[0034] (2) Establishment of stable cell lines: pCD-CMV-MCS-EF1-Puro was used as an overexpression vector, and pLKO.1-puro was used as a knockdown vector. HEK293T cells were used for virus packaging, and the viruses were collected 48 hours later for cell infection. The oligonucleotides of shRNA are shown in Table 2.

[0035] Table 2

[0036] (3) In vitro experimental verification: This example first quantitatively analyzed the infiltration of tumor-associated macrophages (TAMs) in the molecular subtypes of medulloblastoma (MB), and found that the infiltration of TAMs in SHH patients was significantly higher than that in other subtypes. Then, the correlation between TAMs and m6A modification was studied, and it was found that the degree of TAMs infiltration was negatively correlated with the expression of METTL14. Further, it was found that TAMs-derived exosomes can promote the progression of SHH-MB, and it was found that certain microRNAs (miRNAs) in exosomes can reduce the overall m6A modification level in SHH-MB by targeting METTL14. On this basis, by analyzing the m6A methylation group map and gene expression data after M2-EXO treatment, candidate genes were screened, among which only FOXD1 had significant changes at the RNA level after M2-EXO treatment in two SHH-MB cell lines. Further studies found that high expression of FOXD1 was associated with poor prognosis, its mRNA was modified by m6A, and m6A modification affected its expression and stability.

[0037] Furthermore, this example verifies the role of FOXD1 in SHH-MB through in vitro experiments, as follows: The present invention discloses the construction of a stably transfected SHH-MB cell line by knocking down FOXD1 ( Figure 1-2 ) to elucidate whether FOXD1 can serve as an immune regulator affecting the SHH-MB microenvironment. Differential gene expression analysis showed that knockdown of FOXD1 significantly upregulated 2423 genes and downregulated 2363 genes at the RNA expression level ( Figure 3 Among the upregulated genes, 2181 candidate genes were found in cells transfected with both shRNAs ( Figure 4 ). Metascape pathway analysis of these 2181 genes showed that they were enriched in pathways such as "cytokine regulation of the immune system, interferon signaling, and cellular response to cytokine stimulation" ( Figure 5 ). Seven of these genes are related to the recruitment and activation of immune cells ( Figure 3 ), including CXCL10 / 11, IFNL1, CCL5, ISG15, CCL20, and TNFAIP3. Co-expression analysis was performed using the R2 online tool (http: / / r2.amc.nl / ), and the results showed that the RNA levels of these genes were significantly increased after FOXD1 knockdown, while FOXD1 overexpression restored them ( Figure 6 ). The effect of FOXD1 on SHH-MB cytokine secretion was then evaluated by ELISA experiments. In the supernatant of Daoy cells with FOXD1 knockdown, it was observed that the secretion of T cell activation factors including CXCL10 / 11, IFNL1 and CCL5 was significantly increased, while FOXD1 overexpression inhibited this secretion ( Figure 7 Daoy was incubated with M2-EXO to evaluate the expression of inflammatory factors, and Q-PCR results showed that M2-EXO could inhibit the expression of these inflammatory factors. However, when FOXD1 was knocked down, their RNA and protein expression levels could be restored ( Figure 8-9 ).

[0038] Based on the above results, it is speculated that the high expression of FOXD1 in SHH-MB cells (possibly induced by highly infiltrating TAMs) may inhibit the release of chemotactic and inflammatory factors. By intervening in FOXD1 expression, the tumor microenvironment (TME) may be reshaped into a proinflammatory immune microenvironment (IME).

[0039] Example 2 This example further verifies through in vivo experiments in mice that substances that inhibit FOXD1 and substances that inhibit PD-1 can effectively inhibit tumor growth.

[0040] Mouse model experiments: NeuroD2:SmoA1 mice (#008831) purchased from Jackson Laboratory were used as SHH-MB models. Mice were injected with rAAV2 / 9-shFOXD1 and anti-PD-1 antibodies or isotype controls, alone or in combination with anti-PD-1 inhibitors ( Fig.10 ), rAAV2 / 9 was used to demonstrate infection efficiency in the molecular layer (ML), Purkinje cell layer (PC), granule cell layer (GCL), and tumor regions of the cerebellum. All animal care in this disclosure followed ethical guidelines and was approved by the Animal Care Committee of Peking University Health Science Center (Permit No. BCJH0201).

[0041] Tumor progression was monitored by magnetic resonance imaging (MRI) scans, and the results showed that knocking down FOXD1 significantly inhibited tumor growth ( Fig.11 ); combined treatment with PD-1 blockade further reduced tumor volume ( Fig.12 ); however, anti-PD-1 antibody treatment alone had no effect on tumor control, and the combination of shFOXD1 and PD-1 blockade effectively prolonged the survival of SHH-MB mice ( Fig.13 ). It can be seen that knocking down FOXD1 can effectively increase the sensitivity of SHH-MB mice to PD-1 inhibitors.

[0042] Furthermore, to determine the knockdown effect of in situ injection of rAAV2 / 9-shFOXD1, hematoxylin-eosin (H&E) staining was performed to delineate the boundary between the tumor and adjacent tissues. Immunohistochemistry (IHC) staining showed that FOXD1 expression was significantly decreased in the rAAV2 / 9-shFOXD1-treated group compared with the control group. Cytotoxic T cell infiltration in the tumor increased after shFOXD1 treatment, while the enrichment of M2 macrophages was the opposite (e.g., Fig.14 Further IHC staining of CXCL10 / 11 (classic T cell chemokines) showed that shFOXD1 increased the expression of CXCL10 / 11 in tumors compared with the control group ( Fig.14 ), indicating that its ability to recruit CD8+ T cells is enhanced.

[0043] The above in vitro and in vivo results indicate that knocking down FOXD1 reshapes the immune microenvironment (IME) in SHH-MB, recruiting more peripheral CD8+ T cells into tumors, thereby enhancing the effectiveness of PD-1 inhibitors.

[0044] The preferred embodiments of the present disclosure are described in detail above; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure. It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0045] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. Use of the composition in the preparation of a drug for treating medulloblastoma, characterized in that: The composition includes a substance that inhibits FOXD1 and a substance that inhibits PD-1.

2. The use according to claim 1, wherein: The medulloblastoma is SHH type medulloblastoma.

3. The use according to claim 1, wherein: The substance that inhibits FOXD1 includes siRNA targeting FOXD1, shRNA targeting FOXD1, antisense nucleotides targeting FOXD1, antibodies against FOXD1 and small molecule compounds that inhibit FOXD1; the substance that inhibits PD-1 includes siRNA targeting PD-1, shRNA targeting PD-1, antisense nucleotides targeting PD-1, antibodies against PD-1 and small molecule compounds that inhibit PD-1, preferably antibodies against PD-1.

4. The use according to claim 3, wherein: The shRNA targeting FOXD1 includes shFOXD1; the positive and negative strands of the nucleotide sequence of the shFOXD1 are shown in SEQ ID NO.1 and SEQ ID NO.2; CCGGGCCCTTCTCCATCGAGAGCATSEQ ID NO.1, CCGGCGAGGAAACAGACATCGACGTSEQ ID NO.

2.

5. The use according to claim 1, wherein: The dosage form of the drug includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.

6. A drug for treating medulloblastoma, characterized in that: The drugs include substances that inhibit FOXD1 and substances that inhibit PD-1.

7. The drug according to claim 6, wherein The substance that inhibits FOXD1 includes siRNA targeting FOXD1, shRNA targeting FOXD1, antisense nucleotides targeting FOXD1, antibodies against FOXD1 and small molecule compounds that inhibit FOXD1; the substance that inhibits PD-1 includes siRNA targeting PD-1, shRNA targeting PD-1, antisense nucleotides targeting PD-1, antibodies against PD-1 and small molecule compounds that inhibit PD-1, preferably antibodies against PD-1.

8. The drug according to claim 6, wherein The medulloblastoma is SHH type medulloblastoma.

9. The drug according to claim 7, wherein The shRNA targeting FOXD1 includes shFOXD1; the positive and negative strands of the nucleotide sequence of the shFOXD1 are shown in SEQ ID NO.1 and SEQ ID NO.2; CCGGGCCCTTCTCCATCGAGAGCATSEQ ID NO.1, CCGGCGAGGAAACAGACATCGACGTSEQ ID NO.

2.

10. The drug according to claim 6, wherein The dosage form of the drug includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.

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