CN117357530A - Application of PDE3A inhibitor in preparation of medicine for treating acute myeloid leukemia - Google Patents
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- CN117357530A CN117357530A CN202311472567.4A CN202311472567A CN117357530A CN 117357530 A CN117357530 A CN 117357530A CN 202311472567 A CN202311472567 A CN 202311472567A CN 117357530 A CN117357530 A CN 117357530A
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Abstract
The invention discloses an application of a PDE3A inhibitor in preparing a medicament for treating acute myelogenous leukemia, and an application of ANA and IDA in combination in preparing the medicament for treating acute myelogenous leukemia. The present invention discloses the relationship of PDE3A expression to AML prognosis and provides strategies for PDE3A inhibitors in the treatment of AML, as well as novel synergistic strategies for acting on AML treatment by the combination of PDE3A inhibitors and chemotherapeutic agents. The current large-dose chemotherapy brings more toxic and side effects to the infants, also brings heavier economic burden, increases the mortality and abandon rate of the infants, increases the sensitivity of tumor cells to the chemotherapeutic drugs through the targeting drugs, can reduce the dosage of the chemotherapeutic drugs, and reduces the toxic and side effects of the chemotherapeutic drugs to the infants and the economic burden of the families of the infants.
Description
Technical Field
The invention relates to an application of a PDE3A inhibitor in preparing a medicament for treating acute myeloid leukemia, and an application of ANA and IDA in combination in preparing a medicament for treating acute myeloid leukemia, belonging to the field of medicines.
Background
Acute Myeloid Leukemia (AML) is a clinically aggressive and heterogeneous hematological tumor with acquired genetic abnormalities. Current treatments for AML are limited, the overall prognosis for AML remains poor, with only about 40% of young patients (< 60 years) able to achieve long-term survival, with survival rates of 70% for children AML patients taking part in the clinical trial, significantly lower than 85-90% of young people and children with Acute Lymphoblastic Leukemia (ALL) undergoing treatment. An urgent need in the art is to identify new molecular targets and therapeutic strategies to improve overall survival in AML patients.
Platelets exhibit complex interactions with malignant cells, including leukemic blast cells, and play a key role in cancer progression, metastasis, and angiogenesis through different mechanisms. In an in situ pancreatic tumor mouse model, the platelet inhibitor clopidogrel significantly reduced tumor growth. The release of platelet microparticles facilitates the delivery of large amounts of bioactive compounds to cancer cells following internalization, a phenomenon that facilitates AML chemotherapy resistance. Phosphodiesterase 3 (PDE 3) is a member of the phosphodiesterase superfamily, comprising two subtypes, phosphodiesterase 3A (PDE 3A) and phosphodiesterase 3B. PDE3A is a widely characterized cyclic nucleotide phosphodiesterase responsible for catalyzing the hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), coordinating many important intracellular signal transduction and cellular processes. PDE3A is expressed primarily in vascular smooth muscle, platelets, oocytes and heart, playing an important role in platelet aggregation and oocyte maturation. Recent studies report cAMP and cGMP independent functions of PDE3A, which suggest PDE3A is up-regulated in several solid tumors, which are associated with poor prognosis. PDE3A has been implicated in activating inflammatory pathways leading to cancer cell stem cells, by inhibiting the cAMP/PKA pathway. Nevertheless, the precise expression pattern and functional significance of PDE3A in AML is unclear.
The PDE3A inhibitor anagrelide (ANA) is a marketed antithrombotic drug and thrombocytopenic agent for the treatment of thrombocythemia associated with myeloproliferative diseases. ANA treatment reduces circulating platelets by inhibiting the process of megakaryocyte hyperproliferation and differentiation. ANA treatment also inhibits proliferation of cancer cells by inducing apoptosis and cell cycle arrest, the efficacy of which depends on protein levels of PDE3A and SLFN 12. Similarly, the PDE3A inhibitor 6- (4-106 (diethylamino) -3-nitrophenyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one (DNMDP) promotes cancer cell death by stabilizing PDE3A-SLFN12 interactions. The PDE3A inhibitor 17- β -estradiol (E2) induces apoptosis of cancer cells via the PDE3A-SLFN12 complex by promoting intracellular interactions of PDE3A and SLFN 12. PDE3A inhibitors zadaverine selectively inhibit the growth of a variety of tumor cells. These findings highlight the versatile role of PDE3A inhibitors in affecting cancer cell fate and highlight their potential therapeutic applications. However, the exact anticancer role of PDE3A inhibitors in AML remains to be elucidated.
In recent years, the family members of AML patients have been motivated by enhanced chemotherapy cure rates, while long-term toxicity and economic burden remain substantial barriers to the initiation or completion of treatment in these families, combination therapy, or as a key strategy to address this problem. The selective XPO1 inhibitor Selinexor has a synergistic effect with idarubicin in vitro and in vivo. Furthermore, BCL-2 selective inhibitor APG2575 exhibits enhanced chemosensitivity when used in combination with homoharringtonine in AML cell lines, AML patient primary cells and murine models. This progression is relevant to the patient because targeted inhibitors can increase chemosensitivity without a corresponding increase in toxicity and side effects.
Disclosure of Invention
The object of the present invention is to provide a relationship between PDE3A expression and AML prognosis and to provide strategies for PDE3A inhibitors in the treatment of AML, as well as novel synergistic strategies for acting on AML treatment by combination of PDE3A inhibitors and chemotherapeutic agents.
The beneficial effects of the invention are as follows:
aml is a neoplastic disease originating from hematopoietic progenitor cells with long term survival rates far lower than ALL, and new therapeutic targets and interventions are urgently needed. We found that PDE3A high expression correlates with poor AML prognosis, a potentially valuable biomarker and therapeutic target.
ANA is mainly used for the treatment of primary thrombocythemia, and whether it has an effect of treating AML has not been studied yet. We now explore the role of this PDE3A inhibitor in treating PDE3A high expression AML, achieve repositioning of the drug, make new drugs can be applied to clinic as soon as possible, reduce time economic cost, raise security.
3. At present, large-dose chemotherapy brings more toxic and side effects to the infants, also brings heavier economic burden, increases the mortality and abandon rate of the infants, increases the sensitivity of tumor cells to the chemotherapeutic drugs through the targeting drugs, can reduce the dosage of the chemotherapeutic drugs, and reduces the toxic and side effects of the chemotherapeutic drugs to the infants and the economic burden of the families of the infants.
Drawings
Fig. 1: PDE3A is highly expressed in AML cells and is associated with poor prognosis.
(A) Western blot analysis of PDE3A protein levels in primary BMMNCs from AML patients (n=12) and healthy donors (n=6) at the university of sulgzhou affiliated children hospital and normalized to GAPDH. (B) In the sub-childhood hospital at the university of su, the relative mRNA expression levels of PDE3A in primary BMMNCs from AML patients (n=12) and BMMNCs from healthy donors (n=6) were quantified and normalized to GAPDH by qRT-PCR. (C) The boxplot of PDE3A expression in GEPIA dataset compares AML cell lines (n=173) to BMMNC (n=70). (D) The patients were divided into two groups, PDE3A high-expression group (n=111) and PDE3A low-expression group (n=37) based on the amount of PDE3A expression obtained by RNA sequencing analysis. EFS (E) and OS (F) in AML patients at the Suzhou university affiliated children Hospital were stratified according to PDE3A expression. (G) AML patient OS data sets in R2 databases stratify tumor AML according to PDE3A expression levels. (H) fusion gene expression in patients.
Fig. 2: PDE3A inhibitors specifically inhibit the growth of PDE 3A-highly expressing AML cells.
(A) Violin plots of PDE3A expression levels of AML cell lines in GEPIA dataset. (B) Western blot analysis of PDE3A protein levels in AML cell lines and normalization to GAPDH. (C) The relative mRNA expression of PDE3A in AML cell lines was quantified by qRT-PCR and normalized to GAPDH. Measurement of cell viability in leukemic cells treated with corresponding concentrations of PDE3A inhibitors ANA (D), DNMDP (E), nauclefine (F), zardaverine (G), E2 (H),IC was calculated for 72 hours based on drug concentration resulting in 50% cell survival 50 Values. IC of (I-J) PDE3A inhibitors in different AML cell lines 50 Values. (K) Primary BMMNCs from AML patients (n=12) were divided into two groups based on expression of PDE3AmRNA levels, PDE3A high expression group (n=6), PDE3A low expression group (n=6), and cell viability was determined by CCK-8 when treated with 5 μmana for 72 h.
Fig. 3: ANA inhibits proliferation of PDE 3A-highly expressing AML cells by blocking the cell cycle and inhibiting colony forming ability.
(A) Colony formation in HEL and MOLM-16 cells was determined by cell counting, colony formation experiments (scale: 500 μm). (B) Cell cycle distribution of HEL and MOLM-16 cells was determined by flow cytometry analysis of PI staining. (C) Apoptosis of HEL and MOLM-16 cells was determined by flow cytometry analysis of Annexin V-FITC PI staining. (D) HEL and MOLM-16 cells were treated with 1. Mu.M ANA for 72 and 120 hours and then Western blotted to detect changes in the cell cycle associated proteins. (E) HEL cells were transfected with over-expressed luciferase and transplanted into NSG mice (2X 10 per mouse) 6 Cells), 14 days later, mice were treated by gavage with PBS and 5mg/kg ANA for 14 days (days 14-28). (F) Bioluminescence imaging test results of AML xenograft mice at 21 days, 25 days, 29 days, 33 days. (G) Kaplan-Meier survival curves for 2 groups of transplanted mice are shown.
Fig. 4: targeting PDE3A down-regulates drug resistance genes in PDE 3A-expressing AML cells.
(A) Volcanic mapping of normalized gene expression in HEL cells with or without ANA treatment for 72 hours was performed by RNA-seq analysis. (B) Analysis of important genetic features from the corresponding NES by the kyoto gene and genome encyclopedia (KEGG). Heat maps of 1 μm ANA treated HEL cells differentially expressed genes are at "focal adhesion". (C) "Apelin Signal path". (D) "MAPK Signal pathway". (E) "PPAR Signal pathway". (F) Highly enriched, each column represents one sample, each row represents one gene, blue represents down-regulation of the gene, and red represents up-regulation of the gene. (G) HEL and MOLM-16 cells were treated with 1. Mu.M ANA for 72 and 120 hours and then subjected to Western blot analysis to detect changes in the relevant proteins.
Fig. 5: the ANA and IDA combined have obvious synergistic inhibition effect on AML cells.
(A-B) HEL and MOLM-16 were treated with indicated concentrations of ANA and IDA for 72 hours, and then CCK-8 experiments were performed to examine the inhibition of HEL and MOLM-16 by the combination. (C-D) CI diagram of ANA/IDA combination treatment in HEL and MOLM-16. (E-F) HEL and MOLM-16 were treated with ANA and HHT at the indicated concentrations for 72 hours, and then CCK-8 experiments were performed to detect synergistic inhibition. CI diagram of ANA/HHT combination therapy in (G-H) HEL and MOLM-16. (I) Primary BMMNCs of AML patients in PDE3A high expression group (n=6) were assayed for cell survival by CCK-8 when treated with DMSO, 0.005 μm IDA alone, 5 μm ANA alone, 0.005 μm IDA and 5 μm ANA for 72 h.
Fig. 6: the effects of ANA in combination with other clinically usual chemotherapeutics were compared with the effects of ANA in combination with IDA and ANA in combination with HHT.
(A-B) HEL and MOLM-16 were treated with indicated concentrations of ANA and VP-16 for 72h, and then CCK8 experiments were performed to examine the inhibition of HEL and MOLM-16 by the combination.
(C-D) HEL and MOLM-16 were treated with indicated concentrations of ANA and Ara-C for 72h, and then CCK8 experiments were performed to examine the inhibition of HEL and MOLM-16 by the combination.
(E-F) HEL and MOLM-16 were treated with indicated concentrations of ANA and Cladribine for 72h, and then CCK8 experiments were performed to examine the inhibition of HEL and MOLM-16 by the combination.
(G-H) HEL and MOLM-16 were treated with indicated concentrations of ANA and Decistabine for 72H, and then CCK8 experiments were performed to examine the inhibition of HEL and MOLM-16 by the combination. Fig. 7: ANA in combination with IDA synergistically inhibits survival of PDE 3A-expressing AML cells via the focal death pathway.
(A) Volcanic patterns of normalized gene expression in HEL cells treated with or without IDA/ANA combinations were plotted using RNA-seq analysis. (B) KEGG analysis of important gene markers from the corresponding NES. (C) The heat map of HEL cell differential expression genes treated by IDA/ANA is remarkably enriched in the MAPK signal path, each column represents one sample, each row represents one gene, blue represents down-regulation of genes, and red represents up-regulation of genes. (D) Treatment with the IDA/ANA combination significantly enriched the inflammation-associated GSEA signal in the HEL cell transcriptional profile. Normalized enrichment scores NES and P values are shown. (E) Fold change and post-adjustment P values of related differentially expressed genes. (F) HEL and MOLM-16 cells were treated with DMSO, 0.005 μM IDA alone, 1 μM ANA alone, 0.005 μM IDA and 1 μM ANA for 72h, and then subjected to Western blotting to detect changes in the associated proteins. (G-H) ELISA the IL-1 beta and IL-18 levels in supernatants from cell cultures were determined.
Fig. 8: the combination of ANA and IDA produces a synergistic anti-leukemia effect on AML xenograft mouse models.
(A) HEL-luci cells were injected intravenously into NSG mice (2X 10 per mouse 6 Individual cells). After 14 days, mice were treated with PBS, IDA alone (0.5 mg/kg, tail vein injection, days 14-16), ANA alone (5 mg/kg, oral, days 14-28), or combinations. (B) Bioluminescence imaging test results of AML xenograft mice at 23 days, 27 days, 31 days. (C) survival curves for mice of each group (n=7). The p-value is determined using a log rank sum test. (D) Weight and image of representative livers of sacrificed mice. (E) Representative spleen weights and images of sacrificed mice. (F) Immunohistochemical staining of hCD45 in bone marrow, liver and spleen tissue sections of experimental mice. (G) Hematoxylin-eosin staining was performed on histological sections of bone marrow, liver and spleen of experimental mice. (H-N) platelet count, platelet volume, white blood cell count, neutrophil count, monocyte count, lymphocyte count, hemoglobin expression levels in the blood routine of each group of experimental mice.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the accompanying drawings and examples.
The experimental materials used in the following experimental methods are readily available from commercial companies unless otherwise specified. Many modifications may be made by one of ordinary skill in the art without departing from the spirit of the invention, and such modifications are intended to fall within the scope of the invention.
Example 1 overexpression of PDE3A in AML suggests poor prognosis
To explore the potential role of PDE3A in AML, we first analyzed its expression of PDE3A in 12 AML patients and 6 healthy donors, and we found that PDE3A expression levels in bone marrow mononuclear cells (BMMNCs) of AML patients were significantly higher than that of BMMNCs of healthy donors in protein and transcript levels (fig. 1A-B). PDE3A overexpression in AML was demonstrated when we examined PDE3A expression in AML in the GEPIA database (http:// GEPIA. Cancer-pku. Cn/index. Html) (FIG. 1C). These results indicate that PDE3A is significantly overexpressed in AML.
To investigate the relationship between PDE3A expression and patient survival, we subsequently studied the relationship between PDE3A expression and prognosis for AML patients from 148 pediatric AML patients enrolled in a retrospective study conducted at the university of su zhou affiliated children hospital, 10 month 2013 to 9 month 2022. Based on the results of RNA sequencing, the transcriptome expression level of PDE3A in the new diagnostic bone marrow samples was obtained (FIG. 1D). 148 patients were divided into two groups based on PDE3A expression, PDE3A high expression group (n=111) and PDE3A low expression group (n=37). The results showed that there was a significant decrease in event-free survival (EFS) in the PDE3A high-expression group AML patients compared to PDE3A low-expression group patients (fig. 1E), no significant difference in Overall Survival (OS) in both groups of patients, but there was a trend of decrease in the PDE3A high-expression group patients' OS (fig. 1F). We searched the R2 database (https:// hgserver1.Amc. Nl/cgi-bin/R2/main. Cgi), and the results from the database indicated that patients with high PDE3A expression in AML had a lower OS than patients with low PDE3A expression (FIG. 1G). Meanwhile, we found that PDE3A was highly expressed in some specific AML subtypes, such as FLT3-ITD (FIG. 1H). PDE3A has shown potential as a valuable biomarker for diagnosis and prognosis of AML patients.
Overall, studies have shown that PDE3A is overexpressed in AML, and that high expression of PDE3A is closely related to poor prognosis in AML patients.
EXAMPLE 2PDE3A inhibitor ANA specifically inhibits growth of PDE 3A-expressing AML cells in vitro and in vivo
To determine the anti-leukemic function of PDE3A inhibitors, we first studied the expression of PDE3A in AML cell lines in the GEPIA database and found that PDE3A was highly expressed in HEL and MOLM-16 (fig. 2A). We then used reverse transcription polymerase chain reaction (RT-PCR) and Western Blot (Western Blot) to study differences in PDE3A expression in leukemia cell lines, including K562, HL60, HEL, MOLM-16, U937, kasumi-1, MV4-11, NB4, MEG-01, CMK, THP1 and M07E cell lines. In these cell lines we observed a significant increase in PDE3A expression levels in HEL and MOLM-16, over other cell lines at both protein and transcript levels (FIGS. 2B-C). Based on these results, HEL and MOLM-16 were determined as representative high PDE3A expression models for AML for subsequent research work. We have found that the PDE3A inhibitor anagrelide (ANA) specifically inhibits the growth of HEL and MOLM-16 in a concentration dependent manner. After 72 hours of ANA treatment, half inhibition concentrations were reached for HEL and MOLM-16 at 0.8. Mu.M and 1.3. Mu.M, respectively. And ANA was found to have no inhibitory effect on other test AML cell lines expressing low levels of PDE3A (FIG. 2D). We found that other PDE3A inhibitors DNMDP, E2, zardaverine, nauclefine also resulted in a significant decrease in proliferation of HEL and MOLM-16 (FIGS. 2E-H) and IC 50's have been calculated (FIGS. 2I-J).
To verify the anti-leukemic function of ANA in PDE 3A-highly expressed AML, primary cells from AML patients were divided into two groups, PDE 3A-highly expressed group (n=6) and PDE 3A-lowexpressed group (n=6), based on their PDE3A expression at the protein and transcription level, and both groups were treated with 5 μm ANA for 72 hours. Consistent with findings in AML cell lines, ANA significantly inhibited proliferation of primary cells from PDE 3A-high expressing group patients and did not exert an inhibitory effect on primary cells from PDE 3A-low expressing group patients.
Due to sensitivity to ANA, we selected half of the inhibitory concentrations of ANA-treated HEL and MOLM-16 for the next experiment. We found in HEL and MOLM-16 that ANA inhibited colony forming ability (fig. 3A) and inhibited cell cycle by blocking cells in G1 phase (fig. 3B), while apoptosis was hardly affected (fig. 3C). Consistent with these results, expression of cyclin-associated proteins, including cyclin D1, CDK4, C-myc, CDK2 and CDK6, was significantly down-regulated at the protein level in a time-dependent manner after ANA treatment compared to the control group (fig. 3D).
To assess the anti-leukemia activity of ANA in vivo, we established a xenograft model of human AML by tail vein injection of 200 ten thousand luciferase-overexpressing HEL cells into NSG mice (fig. 3E). From day 14 after leukemia cell implantation, we treated mice daily with PBS or 5mg/kg ANA by gavage for 14 days of the whole course. In vivo imaging analysis showed that ANA treatment reduced the leukemia burden of NSG mice compared to PBS group (fig. 3F), and ANA significantly prolonged survival time of NSG mice compared to PBS group (fig. 3G).
It follows that PDE3A inhibitors have the potential to inhibit proliferation of PDE 3A-highly expressing AML cell lines. ANA inhibits the colony forming ability by blocking the cell cycle in the G1 phase, inhibits the leukemia occurrence of a mouse xenograft model, and has anti-leukemia effect on PDE3A high-expression AML cells in vitro and in vivo.
Example 3 targeting PDE3A down-regulates chemotherapeutic resistance genes in PDE3A high expression AML cell lines
To further elucidate the underlying mechanisms of ANA's anti-leukemia function, we performed RNA sequencing analysis on HEL cells, mRNA expression clustering and mapping to identify differentially expressed genes. Overall, ANA treatment for 72 hours resulted in dynamic changes in transcription with 146 genes significantly up-regulated and 197 genes significantly down-regulated (adjust P <0.05, |log2Fold-change| > 0.5) (fig. 4A). The kyoto gene and genome encyclopedia (KEGG) analysis showed a high enrichment of molecules associated with "focal adhesion signaling pathway", "MAPK signaling pathway", "Apelin signaling pathway" and "PPAR signaling pathway" (fig. 4B). Notably, MAPK signaling pathways include ERK1/2, P38, JNK, and ERK5 cascade reactions, which are known to be involved in a variety of biological processes, including metabolic reprogramming, cell proliferation, survival, and differentiation. This knowledge led us to study the effect of ANA treatment on the phosphorylated forms of these key proteins. From the above phenotypic analysis, our studies showed that ANA treatment promoted a time-dependent decrease in protein levels of phosphorylated ERK1/2, p38, JNK and ERK5, which was observed at protein levels by western blot analysis (fig. 4G). At the same time, the generated heat map highlights the significant down-regulation of transcription of cyclin D1 (fig. 4C), similar to the results of previous western blot analysis.
Notably, the heat map depicting the changes in gene expression in the four signaling pathways described above revealed a remarkable observation that genes associated with chemotherapy resistance mechanisms, including PAK1, HMGCS1, p-SRC, DUSP16 and EGR1, exhibited significant downregulation at the transcriptional level (fig. 4C-F). Recent studies report chemotherapy resistance of these genes and underscores the potential to target these chemotherapy resistant genes to enhance cancer chemotherapy sensitivity, western Blot showed that ANA treatment rapidly reduced protein levels of p-SRC, PAK1, HMGCS1, EGR1 and DUSP16 in a time-dependent manner (fig. 4G, figures 0, 3, 5 refer to ANA treatment for 0, 3, 5 days).
In summary, ANA inhibits growth of PDE 3A-highly expressing AML cells by down-regulating MAPK signaling pathways and down-regulating chemotherapy resistance genes, and has the potential to enhance chemotherapy sensitivity.
Example 4 Anagrel in combination with idarubicin has a clear synergistic effect on PDE 3A-highly expressing AML cells
To evaluate whether ANA affects the sensitivity of PDE3A highly expressing AML cells to chemotherapeutic drugs, we screened potent drugs used in combination with ANA, first studied the concentrations of the clinically usual chemotherapeutic drugs Idarubicin (IDA), homoharringtonine (HHT), cytarabine (Ara-C), etoposide (VP-16), decitabine (DEC) and cladribine (Clad) which hardly inhibited cell growth for 72 hours, and then synergistically combined these concentrations of individual anti-leukemia drugs with concentration-dependent ANA to investigate the effect of inhibiting proliferation of HEL and MOLM-16, the results are shown in fig. 5-6. Concurrent IDA treatment with ANA significantly reduced the cell growth rate of HEL and MOLM-16 (FIGS. 5A-B). The CI values at the various doses were almost less than 1 when treated for 72 hours (FIGS. 5C-D). Simultaneous HHT and ANA treatment also reduced the cell growth rate of HEL and MOLM-16. However, HHT combined with ANA was less effective than IDA combined with ANA (FIGS. 5E-H). We found that the combination of IDA and ANA had the strongest synergy among the 6 clinically common anti-leukemia drugs, as judged by the CI index of 50% cytostatic at the ineffective concentration of chemotherapeutic drugs. Furthermore, to verify the effect of the combination in PDE 3A-highly expressing AML cells, primary cells from AML patients were classified into PDE 3A-highly expressing groups (n=6) according to their expression of PDE3A at the transcriptional level. Treatment with vehicle control, vehicle control in DMSO-added medium, 0.005 μm IDA, 5 μm ANA, 0.005 μm IDA, and 5 μm MANA. According to primary cell experiments, combination treatment of IDA and ANA significantly reduced cell viability compared to single drug (fig. 5I). This progression is relevant to the patient because targeted inhibitors can increase chemosensitivity without a corresponding increase in toxicity and side effects.
The ANA in combination with IDA has a synergistic effect on both PDE 3A-highly expressed AML cell lines and primary cells of PDE 3A-highly expressed AML patients.
Example 5 combination of ANA and IDA triggering the cleavage of GSDME to trigger apoptosis and synergistically inhibit the growth of PDE 3A-highly expressing AML cells
To elucidate the complex molecular mechanisms of the synergistic anti-leukemia effects of ANA and IDA, we performed RNA sequencing analysis on HEL cells. Our analysis of the integrated data showed that the combination of ANA and IDA produced significant transcriptional changes within 72 hours, and this combination treatment resulted in significant up-regulation of 906 genes and significant down-regulation of 777 genes (adjust P <0.05, |log2Fold-change| > 0.5) (fig. 7A). KEGG analysis showed a high enrichment of molecules associated with "neutrophil extracellular trap formation", "MAPK signaling pathway", "alcoholism" and "systemic lupus erythematosus" (fig. 7B). Interestingly, recent studies indicate that inhibition of the MAPK signaling pathway can reduce inflammation, a phenomenon that is demonstrated by the results of the study in fig. 4E. Notably, the pro-inflammatory factor IL-1β expression was up-regulated following ANA-in combination IDA treatment (fig. 7C). To verify the observations of the heat map analysis, we performed comprehensive functional annotation and enrichment analysis of differentially expressed genes using the Gene Set Enrichment Analysis (GSEA). The results clearly indicate that the combination treatment of IDA and ANA actively enriches the inflammatory response pathway (fig. 7D).
Given the significantly increased expression levels of inflammatory factors in AML cells, we speculate that the way in which cells die may be pyro-apoptosis, necrotic apoptosis and ubiquity, and RNA sequencing analysis showed little change in genes associated with necrotic apoptosis at the transcriptome level, and altered genes associated with pyro-apoptosis such as GSDME and NLRP3, the inflammatory factor IL-1 β was highly up-regulated at the transcriptome level (fig. 7E). In the present study, consistent with previous RNA sequencing analysis, expression of the apoptosis-related proteins including NLRP3, clear-CASP 8 and GSDME-N was significantly up-regulated with little change in clear-PARP at protein levels when treated with IDA and ANA compared to IDA or ANA alone (FIG. 7F). ELISA analysis showed that IL-1 beta and IL-18 levels in cell culture supernatants were significantly up-regulated when IDA was combined with ANA compared to either IDA or ANA alone (FIG. 7G-H).
In summary, ANA in combination with IDA synergistically inhibits survival of PDE 3A-highly expressing AML cells, the primary mechanism of which is pyrosis caused by GSDME cleavage initiated by Caspase-3 activation.
Example 6 synergistic anti-leukemia effects of ANA and IDA combination on AML xenograft mouse models
Finally, we tested the efficacy of the ANA-IDA combination in vivo using an established AML xenograft mouse model. We injected 200 ten thousand luciferase-overexpressing HEL cells into the tail vein of NSG mice. Mice were closely monitored for clinical symptoms of leukemia. Mice were treated with vehicle control, IDA (0.5 mg/kg), ANA (5 mg/kg), IDA (0.5 mg/kg) in combination with ANA (5 mg/kg) starting at day 14 after leukemia cell injection (FIG. 8A). The mice were monitored until death, and the results of the small animal bioluminescence imaging system showed that the combination treatment of IDA and ANA significantly reduced the mice leukemia burden (fig. 8B) and prolonged the mice survival time (fig. 8C) compared to drug alone treatment. In a second HEL xenograft mouse study, vehicle and drug treated mice were sacrificed, livers and spleens were harvested and weighed, and the IDA-ANA combination treated mice had smaller and lighter livers than the other groups and control groups (fig. 8D-E). The results explain the evidence that IHC analysis of anti-human CD45 cells in liver, spleen and bone marrow of mice following IDA-aNA combination treatment observed a significant reduction in this tumor burden (fig. 8F), as well as a significant reduction in HEL cell histological infiltration in liver, spleen and bone marrow for IDA-aNA combination (fig. 8G), compared to treatment with ANA alone or IDA alone. Blood routine examination found that in addition to thrombocytopenia, a known side effect of ANA and IDA, other types of blood cells were not quantitatively affected, demonstrating the safety of the drug combination (fig. 8H-N).
Under the condition that the signed informed consent and the ethics committee pass the examination, the clinical experiment is carried out on the infant, and the feasibility and the clinical value of the infant are explored. If successful, the recurrence rate and death rate of AML are greatly reduced, and the method has great clinical significance and social value.
Claims (4)
- Use of a pde3a inhibitor in the preparation of a medicament for the treatment of acute myeloid leukemia.
- 2. Use according to claim 1, characterized in that the PDE3A inhibitor is ANA.
- Use of a combination of ana and IDA in the preparation of a medicament for the treatment of acute myeloid leukemia.
- 4. A medicine for treating acute myelogenous leukemia is characterized in that the active ingredients comprise ANA and IDA.
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