A pharmaceutical composition for treating PIK3CA mutation colon cancer and its application

Abstract

This invention belongs to the field of biomedical technology, specifically disclosing a pharmaceutical composition for treating PIK3CA-mutated colorectal cancer and the application of Alpelisib and Menin-MLL complex inhibitors in combination therapy for colorectal cancer. Experimental results show that the combined use of Alpelisib and Menin-MLL 1 / 2 interaction inhibitors can synergistically inhibit the growth of PIK3CA-mutated colorectal cancer PDX tumors and even induce tumor regression, demonstrating better therapeutic effects than Alpelisib alone. The combination of the two has a synergistic therapeutic effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically discloses a pharmaceutical composition for treating PIK3CA mutation colon cancer and application of Alpelisib and Menin-MLL complex inhibitor in a drug for combined treatment of colon cancer. Background Art

[0002] The phosphatidylinositol 3-kinase (PI3K) signaling pathway is one of the top ten signaling pathways in tumors. The PI3K signaling pathway is abnormally active in many tumors and is involved in regulating multiple biological functions such as cell proliferation, differentiation, apoptosis and metastasis. PIK3CA, the gene encoding the catalytic subunit p110α of the PI3K protein complex, is one of the most frequently mutated oncogenes in tumors. Analysis of 11,219 tumor samples of 32 tumor types in TCGA showed that the average mutation rate of PIK3CA was 14%, and its mutation rate in colorectal cancer reached 30%, making it one of the key mutant genes that induce colon cancer. In colorectal cancer, PIK3CA / p110α mutations are mainly concentrated in two hot spots: (1) residues E542, E545 and Q546 in the helical domain (accounting for about 45%); (2) residue H1047 in the kinase domain (accounting for about 30%). Studies have found that PIK3CA hotspot mutations abnormally increase the kinase activity of PI3K, phosphorylate and activate the downstream AKT signaling pathway, leading to tumorigenesis and metastasis. The applicant's completed research has also confirmed the important role of PIK3CA helical domain hotspot mutations in the occurrence and development of colon cancer. However, at present, research on the carcinogenic mechanism of PIK3CA mutations mainly focuses on its regulation of the mutant cells themselves, and whether the carcinogenic mutation affects the biological function and molecular mechanism of colon epithelial cells remains to be revealed. Therefore, in-depth research on the mechanism of tumorigenesis caused by PIK3CA mutations is important for a comprehensive understanding of the function of PI3K in tumors and exploring potential effective PI3K targeted treatment options.

[0003] PIK3CA / p110α is one of the key targets for cancer treatment. The earliest generation of small molecule inhibitors of PI3K is called Pan-PI3K inhibitor. Because this type of inhibitor can simultaneously inhibit multiple isoforms of PI3K, including p110α (encoded by the PIK3CA gene), p110β (encoded by the PIK3CB gene), p110γ (encoded by the PIK3CG gene), and p110δ (encoded by the PIK3CD gene), there are greater toxic side effects and off-target risks, and the patient's tolerance and treatment effect are not ideal. Therefore, how to design a more effective treatment plan for PIK3CA mutant tumors requires further research. Summary of the invention

[0004] The present invention provides a pharmaceutical composition for treating PIK3CA mutation colon cancer and application of Alpelisib and a Menin-MLL complex inhibitor in a drug for combined treatment of colon cancer.

[0005] In one aspect, the present invention provides a pharmaceutical composition comprising a combination of an Alpelisib inhibitor and a Menin-MLL inhibitor.

[0006] The Alpelisib inhibitor has a structure as shown in formula (1), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, geometric isomer or tautomer thereof;

[0007]

[0008] The Menin-MLL inhibitor has a structure as shown in formula (2), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, geometric isomer or tautomer thereof, wherein R in formula (2) is a chain alkyl group having 1 to 3 carbon atoms;

[0009]

[0010] In one embodiment of the present invention, in formula (2), R is methyl or ethyl, preferably methyl.

[0011] In one embodiment of the present invention, the mass ratio of the Alpelisib inhibitor to the Menin-MLL inhibitor is 2:1.

[0012] In one embodiment of the present invention, the Alpelisib inhibitor is selected from BYL719.

[0013] In one embodiment of the present invention, the Menin-MLL inhibitor is selected from VTP50469.

[0014] On the other hand, the present invention provides the use of Alpelisib and a Menin-MLL complex inhibitor in a drug for the combined treatment of PIK3CA mutant colon cancer.

[0015] The pharmaceutical composition is used for preparing a drug for inhibiting the growth of colon cancer cells and inducing apoptosis of colon cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 . Schematic diagram of the distal acclimation model demonstrating that PIK3CA mutant tumor cells induce malignant transformation of colon epithelial cells;

[0017] Figure 2. Schematic diagram of the study on non-targeted metabolomics mass spectrometry detection of differential metabolites in donor cells, exosomes, exosome-free supernatants and recipient cells;

[0018] Figure 3 . Schematic diagram of the study on the interaction between AA and Menin protein to regulate the function of Compass complex;

[0019] Figure 4 . Schematic diagram of the progress of combined blockade of PIK3CA mutation "inside and outside" oncogenic signaling in the treatment of colon cancer PDX models. DETAILED DESCRIPTION

[0020] Among all human cancers, the PIK3CA oncogene is one of the genes with the highest mutation rate. Previous studies have revealed the carcinogenic mechanism of this gene mutation "inside" colon cancer cells. However, it is still unknown whether PIK3CA mutant tumor cells affect the functional activity of normal cells and promote the malignant progression of colon cancer.

[0021] In order to reveal this scientific problem, the applicant of the present invention inoculated tumor cells and NCM460 cells on both sides of nude mice, such as Figure 1 As shown in A, in the figure, A. Schematic diagram of the remote domestication model; BE. 106 PIK3CA mutant (or wild-type) tumor cells were randomly inoculated on one side of the nude mouse, and 107 NCM460 cells were inoculated on the other side of the nude mouse. The incidence and growth rate of subcutaneous tumors were statistically analyzed, and the tumor growth curve was drawn; F. The subcutaneous tumor was taken, and the tumor cells and colon epithelial cells were labeled with GFP antibodies and mCherry antibodies, respectively, to observe whether there were tumor cells (green) in the subcutaneous tumor (red) formed by NCM460 cells; GH. The subcutaneous tumor formed by NCM460 cells was removed, cut into small pieces, and continued to be inoculated subcutaneously into nude mice to construct the NCM460 F2 XDX model, and the incidence and growth rate of subcutaneous tumors were statistically analyzed, and the tumor growth curve was drawn. The results showed that PIK3CA mutant tumor cells can remotely induce non-tumorigenic NCM460 cells to form subcutaneous tumors visible to the naked eye, while PIK3CA WT tumor cells do not have this induction ability ( Figure 1 BE). Moreover, no PIK3CA mutant tumor cells were detected in the subcutaneous tumor tissue formed by NCM460 cells, suggesting that this subcutaneous tumor was indeed formed by the malignant transformation of NCM460 cells themselves ( Figure 1F). The applicant further removed the subcutaneous tumor formed by NCM460 cells, cut it into small pieces, and continued to inoculate it subcutaneously into nude mice to form the NCM460 F2 XDX (Xenograft derived xenograft) model. The results showed that NCM460 F2 XDX showed almost the same tumor incidence and tumor growth curve as the subcutaneous tumor model formed by wild-type tumor cells ( Figure 1 G and H). This suggests that PIK3CA mutant tumor cells can remotely induce normal colon epithelial cells to undergo malignant transformation and become malignant tumor cells. PIK3CA mutant tumor cells have the ability to transform colon epithelial cells into tumor cells through a paracrine mechanism.

[0022] At the same time, in recent years, exosomes have been confirmed to be involved in the intercellular signal transduction process and are key messengers mediating intercellular signal transmission. In order to clarify the key mediators of exosomes that transmit carcinogenic functions, the applicant conducted non-targeted metabolomics mass spectrometry detection on donor tumor cells, tumor cell-derived exosomes, exosome-free supernatants, and colon epithelial cells stimulated by supernatants, such as Figure 2 As shown in the figure, A and C. Non-targeted metabolomics mass spectrometry detection of differential metabolites in PIK3CA mutant and wild-type tumor cells (donor cells), exosomes, exosome-free supernatants and colon epithelial NCM460 cells (recipient cells). B and D. Arachidonic acid targeted quantitative mass spectrometry detection and analysis of AA levels in PIK3CA mutant and wild-type tumor cells (donor cells), exosomes, exosome-free supernatants and colon epithelial NCM460 cells (recipient cells). The results showed that compared with wild-type tumor cells, arachidonic acid (AA) levels in PIK3CA mutant tumor cells were more than 1000 times higher ( Figure 2 A and 2C, left); The difference in AA content in exosomes is more obvious. Compared with wild-type cell-derived exosomes, AA in mutant cell-derived exosomes is more than 5000 times higher ( Figure 2 A and 2C, second from left), indicating the enrichment of exosomes; while in the exosome-free supernatant, there was almost no AA ( Figure 2 A and 2C, second from the right); in colon epithelial cells stimulated by exosomes, the AA level in colon epithelial NCM460 cells stimulated by mutant tumor cell-derived exosomes increased by nearly 1000 times compared with that stimulated by wild-type tumor cell-derived exosomes ( Figure 2A and 2C, right). Further quantitative mass spectrometry detection of arachidonic acid revealed that arachidonic acid was significantly upregulated in the supernatant of mutant tumor cells and in exosomes derived from the supernatant of mutant tumor cells compared with wild-type tumor cells ( Figure 2 B and 2D, upper left and upper right); the content of arachidonic acid in the exosome-free supernatant was extremely low, and there was no statistical difference between the groups ( Figure 2 B and 2D, lower left); in colon epithelial cells treated with mutant tumor cell-derived exosomes, the content of arachidonic acid was significantly higher than that in NCM cells treated with wild-type tumor cell-derived exosomes ( Figure 2 B and 2D, lower right). The above metabolic mass spectrometry results suggest that arachidonic acid is a key "signal mediator" for PIK3CA mutant cells to transmit oncogenic functions. PIK3CA mutant cells induce malignant transformation of colon epithelial cells through exosome-mediated arachidonic acid (AA).

[0023] As an important epigenetic regulatory mechanism, histone methylation modification is undoubtedly an important link in the occurrence and development of tumors. Therefore, the regulatory effect of arachidonic acid on histone methylation was first detected. Figure 3 As shown in the figure, A. Exogenous AA stimulated NCM cells for 48 hours, total cell protein was extracted, and WB was used to detect the levels of histone H3K4me3, H3K9me3, H3K27me3, H3K36me3, and H3K79me3. B. Exogenous AA stimulated NCM cells for 48 hours, total cell protein was extracted, and WB was used to detect the protein expression levels of different component subunits in the Compass complex. C. Molecular docking simulated the interaction region between AA and Menin protein and predicted potential binding sites. D. Microscale thermophoresis (MST) was used to detect the interaction between AA and Menin protein. E. NCM cells were treated with AA stimulation and AA stimulation combined with VTP50469, the cells were lysed, and IP experiments were performed with Menin antibodies. WB was used to detect the effects of different treatments on the interaction between Menin and MLL1 / 2. F. Schematic diagram of the "mortise and tenon structure" model of the interaction between Menin, AA and MLL1 / 2. WB results showed that arachidonic acid stimulation specifically caused an increase in the level of histone H3K4 trimethylation (H3K4me3) in colon epithelial cells ( Figure 3 A).

[0024] Then we explored the molecular mechanism of AA regulating H3K4me3 formation. Compass complex members play an important role in establishing H3K4 methylation modifications in different regions of chromatin. First, we detected the expression levels of each subunit in the Compass complex. WB results showed that arachidonic acid did not affect the protein levels of these subunits in the Compass complex ( Figure 3 B). Recent studies have found that the regulatory effects of small molecule metabolites in cell functions are often achieved by targeting interacting proteins. Therefore, the applicant speculates that AA may affect the functional activity of the Compass complex by interacting with a subunit in the Compass complex, thereby promoting the expression level of H3K4me3. In order to find the interacting proteins of AA, the applicant used a molecular docking model to find that AA can directly bind to the regulatory subunit Menin protein. The simulation results showed that the binding force between AA and Menin is positive, and the 323rd tyrosine (Tyr323) of the Menin protein may be the key hydrogen bond binding site for its interaction with the small molecule metabolite AA ( Figure 3 C). Microscale thermophoresis (MST) is an important method to characterize the interaction between biological molecules. The results of MST experiments further confirmed that AA interacts with Menin protein ( Figure 3 D). The above results fully demonstrate that Menin, the regulatory subunit of the Compass complex, may be an interacting protein of AA.

[0025] We further explored how AA regulates the function of the Compass complex by interacting with the Menin protein. The Menin-MLL1 / 2 interaction is a key factor in regulating the activity of MLL methyltransferase and thus affecting its gene transcription regulation function. Therefore, the applicant speculates that the interaction between AA and Menin may promote or activate the binding of the latter to MLL methyltransferase. At the same time, VTP50469 was used as a Menin-MLL inhibitor for IP experiments. The experimental results showed that AA stimulation did promote the binding of Menin to MLL1 / 2 ( Figure 3 E). VTP50469 is a new drug developed in 2019 to treat hematological tumors by targeting the Menin-MLL interaction. The inhibitor dissociates Menin protein from MLL by targeting it. Coincidentally, Tyr323 (AA-Menin interaction site predicted by molecular docking) is one of the key sites for VTP50469 to bind to Menin. The results of immunoprecipitation experiments also showed that VTP50469 almost completely blocked the AA-induced Menin-MLL interaction ( Figure 3 E). Therefore, the applicant speculates that AA may bind to the target protein Menin through the Tyr323 site, mediating the binding of Menin-MLL. The interaction mode of Menin-AA-MLL can be figuratively compared to the "mortise and tenon structure" model that maintains overall stability in ancient Chinese wooden buildings ( Figure 3 F) AA can significantly stabilize the Menin-MLL binding, while the small molecule inhibitor VTP50469 can block the effect of AA, demonstrating its "potential" for treating colon cancer patients with high AA levels.

[0026] The following describes the implementation of the present invention.

[0027] Previous studies have shown that PIK3CA mutant tumor cells parasecrete exosomal arachidonic acid (AA) to colon epithelial cells, and then AA directly binds to Menin protein, promoting the interaction between Menin and MLL1 / 2 methyltransferase complex, activating the expression of downstream target genes in an epigenetic reprogramming manner. Blocking the interaction between AA and Menin protein indicates the failure of the signal transduction pathway of PIK3CA mutant tumor cells to induce malignant transformation of intestinal epithelial cells.

[0028] Previous studies have found that mutant tumor cells have both "internal" and "external" carcinogenic signaling pathways. Therefore, the applicant proposed a new scheme to treat colon cancer by simultaneously cutting off the "internal and external" carcinogenic pathways of mutant tumor cells. Specifically, the applicant used BYL719 as an Alpelisib inhibitor and VTP50469 as a Menin-MLL inhibitor, with a dosage ratio of 2:1, and the dosage was: Alpelisib / BYL719 was 12.5 mg / kg, and VTP50469 was 6.25 mg / kg, and functional experiments were conducted in mice. Figure 4 As shown, the applicant used an "internal" pathway inhibitor (PIK3CA inhibitor Alpelisib), an "external" pathway inhibitor (Menin-MLL interaction inhibitor) and a combination of the two monotherapy to treat colon cancer PDX models ( Figure 4 A).

[0029] Primary human cancer tumor tissues of 2 PIK3CA WT (2 cases) or PIK3CA mutants (E545K and H1047R) were inoculated subcutaneously into 6-week-old female immunodeficient BALB / c nude mice. For drug treatment, Alpelisib was dissolved in a solvent solution containing 5% DMSO, 50% PEG-300, 5% Tween-80, and 40% ddH2O. VTP50469 was dissolved in corn oil containing 5% DMSO. When the PDX tumors reached a size of 100-150 mm3, the mice were randomly divided into four groups (8 mice in each group): vehicle group, Alpelisib group (12.5 mg / kg, oral gavage, once a day), VTP50469 group (50 mg / kg, oral gavage, once a day), and Alpelisib plus VTP50469 combination group (12.5 mg / kg Alpelisib combined with 50 mg / kg VTP50469). Figure 4A. Construction of human colon cancer PDX model and combined treatment scheme. BE. Using PIK3CA wild-type (2 cases) and mutant (2 cases) PDX models, the "internal" pathway inhibitor (PIK3CA inhibitor Alpelisib), "external" pathway inhibitor (#1) and the combination of the two single drugs were used to treat the PDX models, and the subcutaneous tumor growth rate (B and C) and mouse body weight (D and E) were recorded. F. Calculation of the synergistic therapeutic effect of Alpelisib and VTP50469 in colon cancer PDX models.

[0030] The results showed that in the PIK3CA wild-type PDX model, the combination of Alpelisib and the Menin-MLL interaction inhibitor VTP50469 did not show a more significant tumor inhibitory effect than either drug alone ( Figure 4 B); however, in PIK3CA mutant tumor cells, combined targeted therapy showed significant synergistic anti-tumor effects, and some subcutaneous tumors shrank or even disappeared in the later stage of combined treatment. Alpelisib and VTP50469 also showed significant synergistic therapeutic effects in the treatment of PIK3CA mutant colon cancer PDX models ( Figure 4 C). It is worth noting that the combined use of drugs did not cause weight loss in mice, suggesting that the toxic side effects of the combined use of drugs were relatively small ( Figure 4 D and 4E). In summary, the strategy of combining Alpelisib and Menin-MLL complex inhibitors to treat PIK3CA-mutated colon cancer is feasible.

[0031] The present study found that PIK3CA mutant tumor cells paracrine arachidonic acid to colon epithelial cells; then arachidonic acid directly binds to Menin protein and promotes Menin-MLL1 / 2 interaction, activating histone H3K4me3 levels, and inducing malignant transformation of colon epithelial cells in a manner of epigenetic reprogramming. The combined use of Alpelisib and Menin-MLL1 / 2 interaction inhibitor can synergistically inhibit the growth of PIK3CA mutant colon cancer PDX tumors and even cause tumor regression.

[0032] The present invention solves the clinical problem that "the use of the PIK3CA mutation-specific inhibitor Alpelisib alone is ineffective in treating colon cancer". The combined use of Alpelisib and Menin-MLL1 / 2 interaction inhibitor can significantly inhibit the growth of PIK3CA mutant colon cancer PDX tumors, showing better therapeutic effects than Alpelisib alone, and the combination of the two has a synergistic therapeutic effect.

[0033] The above disclosure is only a specific embodiment of the present invention, but the embodiments of the present invention are not limited thereto, and any changes that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for treating PIK3CA mutation colon cancer, characterized in that: The pharmaceutical composition is a combination of an Alpelisib inhibitor and a Menin-MLL inhibitor, the Alpelisib inhibitor is BYL719, the Menin-MLL inhibitor is VTP50469, and the mass ratio of the Alpelisib inhibitor to the Menin-MLL inhibitor is 2:

1.

2. Use of the pharmaceutical composition according to claim 1 in the preparation of a drug for treating PIK3CA mutation colon cancer.

3. The use according to claim 2, characterized in that: The pharmaceutical composition is used for preparing a drug for inhibiting the growth of colon cancer cells and inducing apoptosis of colon cancer cells.

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