Apoptosis In Acute Myeloid Leukemia Research Articles

Structure-Activity Relationship of Hydroxycinnamic Acid Derivatives for Cooperating with Carnosic Acid and Calcitriol in Acute Myeloid Leukemia Cells.

Plant phenolic compounds have shown the ability to cooperate with one another at low doses in producing enhanced anticancer effects. This may overcome the limitations (e.g., poor bioavailability and high-dose toxicity) in developing these agents as cancer medicines. We have previously reported that the hydroxycinnamic acid derivative (HCAD) methyl-4-hydroxycinnamate and the phenolic diterpene carnosic acid (CA) can synergistically induce massive calcium-dependent apoptosis in acute myeloid leukemia (AML) at non-cytotoxic concentrations of each agent. Here, we explored the chemical nature of the synergy between HCADs and either CA, in inducing cytotoxicity, or the active metabolite of vitamin D (calcitriol), in enhancing the differentiation of AML cells. This was done by determining the structure–activity relationship of a series of hydroxycinnamic acids and their derivatives (methyl hydroxycinnamates and hydroxybenzylideneacetones) in combination with CA or calcitriol. The HCAD/CA synergy required the following critical structural elements of an HCAD molecule: (a) the para-hydroxyl on the phenolic ring, (b) the carbon C7–C8 double bond, and (c) the methyl-esterified carboxyl. Thus, the only HCADs capable of synergizing with CA were found to be methyl-4-hydroxycinnamate and methyl ferulate, which also most potently enhanced calcitriol-induced cell differentiation. Notably, the C7–C8 double bond was the major requirement for this HCAD/calcitriol cooperation. Our findings may contribute to the rational design of novel synergistically acting AML drugs based on prototype combinations of HCADs with other agents studied here.

An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML.

CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. SIGNIFICANCE: There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 275.

MiR-335-3p inhibits cell proliferation and induces cell cycle arrest and apoptosis in acute myeloid leukemia by targeting EIF3E.

Here, we aimed to investigate the biological roles and the regulatory mechanisms of miR-335-3p in acute myeloid leukemia (AML). We first found miR-335-3p was significantly downregulated in blood samples from leukemia patients and cell lines using reverse transcription quantitative polymerase chain reaction. Through CCK-8 assay and flow cytometry, we observed that miR-335-3p overexpression significantly inhibited cell proliferation and induced cell cycle G0/G1 arrest and apoptosis in AML cell lines (THP-1 and U937). Moreover, miR-335-3p directly targets EIF3E and negatively regulated its expression. More importantly, EIF3E overexpression reversed the effects of miR-335-3p on cell proliferation, G1/S transition, and apoptosis. Furthermore, miR-335-3p overexpression obviously downregulated the expression of CDK4, Cyclin D1, and Bcl-2, while upregulated the expression of p21 and Bad, which were significantly rescued by the cotransfection of pcDNA3.1-EIF3E. Collectively, our study proposes that miR-335-3p/EIF3E axis could be a promising therapeutic target to mitigate the progression of AML.

Effect of rhTPO to the Proliferation and Apoptosis of Acute Myeloid Leukemia Cell Lines

To investigate the effects of recombinant human thrombopoietin (rhTPO) to proliferation and apoptosis of acute myeloid leukemia (AML) cell lines. After the treatment of different concentrations of rhTPO (0, 50, 100 ng/ml) for different time (24,48,72 h)，the cell proliferation rates of the AML cell lines (Kasumi-1, Skno-1, HEL, HL-60, THP-1) were determined by CCK-8 method. Apoptosis rate of each cell line cocultured with rhTPO was detected by Annexin V/PI method. The relative expression of TPO receptor c-MPL (myeloproliferative clonal antibody) mRNA in AML cell lines was detected by Q-PCR. The expression of c-MPL protein in each cell line was detected by Western blot. The expression of c-MPL antigen in HL-60 cells treated by different concentrations of rhTPO was detected by Flow cytometry. RhTPO showed no promotion to the proliferation of Kasumi-1, Skno-1, HEL, HL-60, THP-1 cell lines，however，it showed inhibitory effect to cell proliferation (72 h 0 ng/ml vs 100 ng/ml, P= 0.029) and pro-apoptotic (48 h 0 ng/ml vs 50 ng/ml, P=0.0143) in HL-60 cells. In Kasumi-1, Skno-1, HEL and THP-1 cells, there showed no statistically significant differences in apoptosis rate among each groups treated by different concentrations of rhTPO. Each AML cell line showed different levels of c-MPL gene and c-MPL protein expression, but HEL cells showed the highest expression in both of them. After HL-60 cells were treated by different concentrations of rhTPO for 48 hours, there showed no statistical difference in c-MPL antigen expression among each groups. RhTPO can not promote the proliferation of Kasumi-1, Skno-1, HEL, HL-60 and THP-1 leukemia cell lines. On the contrary, rhTPO can inhibit HL-60 cell proliferation and promote its apoptosis, and this effect is not related to c-MPL gene expression or protein expression.

Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia.

Simple SummaryAcute Myeloid Leukemia is an aggressive disease with poor outcomes. New targeted therapies that can boost the effects of currently used chemotherapy medications without added toxicity are needed. Targeting an overactive kinase, called the protein Kinase CK2 in AML, helps leukemia cells undergo cell death and helps certain chemotherapy drugs work better. Here, we present evidence that CX-4945, a CK2 inhibitor drug, effectively kills leukemia cells in mouse models and shows the mechanism of action responsible for these effects. Leukemia cells are more sensitive to a decrease in CK2 kinase levels than normal cells. Our results show that inhibiting CK2 kinase makes AML cells more susceptible to anthracycline-induced cell death. Anthracyclines like daunorubicin and doxorubicin are widely used to treat leukemia in children and adults. A rational combination of protein kinase CK2 inhibitors with the standard of care chemotherapy may help treat AML more effectively.Protein Kinase CK2 (Casein Kinase 2 or CK2) is a constitutively active serine-threonine kinase overactive in human malignancies. Increased expression and activity of CK2 in Acute Myeloid Leukemia (AML) is associated with a poor outcome. CK2 promotes AML cell survival by impinging on multiple oncogenic signaling pathways. The selective small-molecule CK2 inhibitor CX-4945 has shown in vitro cytotoxicity in AML. Here, we report that CX-4945 has a strong in vivo therapeutic effect in preclinical models of AML. The analysis of genome-wide DNA-binding and gene expression in CX-4945 treated AML cells shows that one mechanism, by which CK2 inhibition exerts a therapeutic effect in AML, involves the revival of IKAROS tumor suppressor function. CK2 phosphorylates IKAROS and disrupts IKAROS’ transcriptional activity by impairing DNA-binding and association with chromatin modifiers. Here, we demonstrate that CK2 inhibition decreases IKAROS phosphorylation and restores IKAROS binding to DNA. Further functional experiments show that IKAROS negatively regulates the transcription of anti-apoptotic genes, including BCL-XL (B cell Lymphoma like–2 like 1, BCL2L1). CX-4945 restitutes the IKAROS-mediated repression of BCL-XL in vivo and sensitizes AML cells to apoptosis. Using CX-4945, alongside the cytotoxic chemotherapeutic drug daunorubicin, augments BCL-XL suppression and AML cell apoptosis. Overall, these results establish the in vivo therapeutic efficacy of CX-4945 in AML preclinical models and determine the role of CK2 and IKAROS in regulating apoptosis in AML. Furthermore, our study provides functional and mechanistic bases for the addition of CK2 inhibitors to AML therapy.

The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia.

Simple SummaryIn this study, we investigated and boosted the pro-apoptotic and pro-differentiating activity of MEDS433 in acute myeloid leukemia (AML). MEDS433 is an inhibitor of Dihydroorotate Dehydrogenase, a fundamental enzyme in the de novo pyrimidine biosynthesis. We discovered that MEDS433 alone and in combination with classical antileukemic agents had a good apoptotic activity, but it could be reduced in vivo due to the physiological presence of uridine. On the contrary, the combination of MEDS433 and dipyridamole, a blocker of the pyrimidine salvage pathway, induced metabolic lethality and myeloid differentiation in all our AML models, while being characterized by a limited toxicity on non-AML cells.Dihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition can induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors had shown promising in vitro and in vivo activity on solid tumors, but their effectiveness was not confirmed in clinical trials, probably because cancer cells exploited the pyrimidine salvage pathway to survive. Here, we investigated the antileukemic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we mimicked human conditions (performing experiments in the presence of physiological uridine plasma levels) and looked for synergic combinations to boost apoptosis, including classical antileukemic drugs and dipyridamole, a blocker of the pyrimidine salvage pathway. MEDS433 induced apoptosis in multiple AML cell lines, not only as a consequence of differentiation, but also directly. Its combination with antileukemic agents further increased the apoptotic rate, but when experiments were performed in the presence of physiological uridine concentrations, results were less impressive. Conversely, the combination of MEDS433 with dipyridamole induced metabolic lethality and differentiation in all AML cell lines; this extraordinary synergism was confirmed on AML primary cells with different genetic backgrounds and was unaffected by physiological uridine concentrations, predicting in human activity.

Effect of MiR-29b-3p Targeting STAT3 on Proliferation and Apoptosis of Acute Myeloid Leukemia Cells

To investigate the effect of miR-29b-3p on apoptosis and proliferation of acute myeloid leukemia (AML) cells by targeting signal transducer and activator of transcription 3 (STAT3). TargetScan and miRanda online databases were used to predict the binding sites of miR-29b-3p and STAT3 3'UTR. The targeting relationship between them was estimated by Dual-Luciferase reporter assay experiment. After miR-29b-3p over-expression, qPCR and Western blot were used to detect the expression of STAT3 mRNA and proteins, flow cytometry to determine the apoptosis of AML cells, and MTS to detect the changes of cell proliferation in each group. Dual-Luciferase reporter assay confirmed that STAT3 was the target gene of miR-29b-3p. After miR-29b-3p overexpression, the expression of STAT3 mRNA and protein decreased. Compared with the control groups, the proliferation of AML cells in the overexpression group decreased and the apoptosis increased (P<0.05). MiR-29b-3p can inhibit the proliferation and induce apoptosis of AML cells by down-regulating STAT3.

Inhibition of EZH1 and EZH2 Restores Chemosensitivity of Leukemia Stem Cells in Acute Myeloid Leukemia By Recruitment of Quiescent AML Stem/Progenitor Cells

Inhibition of EZH1 and EZH2 Restores Chemosensitivity of Leukemia Stem Cells in Acute Myeloid Leukemia By Recruitment of Quiescent AML Stem/Progenitor Cells

A Synthetic Lethal Approach to Eradicate AML Via Synergistic Activation of Pro-Apoptotic p53 By MDM2 and BET Inhibitors

Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually compared to many other cancers, over 90% of AML patients retain wild type TP53, encoding pro-apoptotic tumor suppressor p53. However, wild-type p53 functions are frequently suppressed by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. MDM2 inhibitors (MDM2i), which activate wild-type p53, show encouraging pre-clinical activity, but limited clinical activity. In an effort to find targets that synergize with p53 activation via MDM2i and minimize toxicity, we performed a cell-based synthetic lethal drug screen and a CRISPR viability screen. These screens identified BRD4 inhibition as a candidate synthetic lethal partner of MDM2i. BRD4 is a member of the Bromodomains and Extraterminal (BET) family of proteins, a transcriptional co-activator and already a candidate AML therapeutic target. Surprisingly, we found inhibition of BRD4 alone induces expression of some of p53 target genes. We unexpectedly reveal that BRD4 binds to p53 target genes and acts as a transcriptional repressor of these genes. Synergistic cell killing by the drug combination (MDM2i + BET inhibitor (BETi)) depends on synergistic activation of p53 target genes, such as PUMA and NOXA, due to simultaneous stabilization of p53 by MDM2i and relief of BRD4-mediated repression by BETi. Our combined therapy of MDM2i and BETi is synergistically lethal to human AML cell lines harboring wild type TP53in vitro, against two mouse models of AML in vivo, and against primary human patient blasts in vitro. Furthermore, we used BET PROTACs to selectively and completely induce degradation of BRD4 in cells. Consistent with results from BETi, BET degraders and MDM2i synergize to suppress cell viability with superior potency. Taken together, our data show BRD4 represses p53-mediated transcription activation and apoptosis in AML. Therefore, co-targeting wild-type TP53 and a transcriptional repressor function of BRD4 represent a novel synthetic lethal vulnerability in AML. Disclosures Latif: AbbVie: Consultancy, Honoraria; Takeda UK: Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Kite: Consultancy, Honoraria, Speakers Bureau; Jazz: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria. Higgins:Roche: Current Employment, Current equity holder in publicly-traded company, Other: Support of parent study and funding of editorial support. Copland:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cyclacel Ltd: Research Funding; Roche: Research Funding; Epizyme: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; Gilead: Speakers Bureau.

Zella 201: A Biomarker-Guided Phase II Study of Alvocidib Followed By Cytarabine and Mitoxantrone in MCL-1 Dependent Acute Myeloid Leukemia (AML): Results of Newly Diagnosed High-Risk Exploratory Arm

Zella 201: A Biomarker-Guided Phase II Study of Alvocidib Followed By Cytarabine and Mitoxantrone in MCL-1 Dependent Acute Myeloid Leukemia (AML): Results of Newly Diagnosed High-Risk Exploratory Arm

HMGB1: an important regulator of myeloid differentiation and acute myeloid leukemia as well as a promising therapeutic target

High mobility group box 1 (HMGB1) is a non-histone nuclear protein which has been intensively studied in various physiological and pathological processes including leukemia. Here in this study, we further demonstrated that HMGB1 presents higher expression in the bone marrow mononuclear cells of acute myeloid leukemia (AML) patients compared with the normal controls and contributes to the AML pathogenesis and progression by inhibiting apoptosis, facilitating proliferation, and inducing myeloid differentiation blockade of AML cells. Mechanistic investigation revealed that transforming growth factor beta-induced (TGFBI) acts as a potential downstream target of HMGB1 and lentivirus-mediated knockdown of TGFBI expression impaired phorbol-12-myristate-13-acetate (PMA) and all-trans retinoic acid (ATRA)–induced myeloid differentiation of AML cell lines. On the other hand, chidamide, an orally histone deacetylase inhibitor, decreases HMGB1 expression significantly in AML cells with concomitant upregulation of TGFBI expression, and confers therapeutic effect on AML by inducing cell differentiation, apoptosis and inhibiting cell proliferation. In conclusion, our findings provide additional insights that HMGB1 is a promising therapeutic target of AML, and also present experimental evidence for the clinical application of chidamide as a novel agent in AML therapy by downregulating HMGB1 expression.Key messagesHMGB1 induces cell proliferation and myeloid differentiation blockade and inhibits apoptosis of AML cells.TGFBI acts as a potential target of HMGB1.Chidamide, a selective HDAC inhibitor, confers promising therapeutic effect for AML via downregulating HMGB1 expression.

Synthesis and biological activity of imidazole group-substituted arylaminopyrimidines (IAAPs) as potent BTK inhibitors against B-cell lymphoma and AML

Bruton’s tyrosine kinase (BTK) is a member of the Tec kinase family and plays a key role in the modulation of the B-cell receptor (BCR)-mediated signaling pathway. Inhibition of BTK has been proven to be an effective therapeutic approach for various hematological malignancies, such as chronic lymphocytic leukemia (CLL), mantle cell leukemia (MCL), diffuse large B-cell lymphoma (DLBCL) and acute myeloid leukemia (AML). Here, a new series of imidazole group-substituted arylaminopyrimidines (IAAPs) were designed and synthesized as potent inhibitors of the enzymatic activity of BTK with a half maximal inhibitory concentration (IC50) ranging from 13.10 to 42.40 nM. In particular, 11a and 11b exhibited stronger antiproliferative activity against AML and B lymphomas cell lines compared with BTK inhibitor ibrutinib and showed low cytotoxicity against normal peripheral blood mononuclear cells (PBMCs). In addition, analysis of the mechanism of action of these compounds revealed that 11a and 11b induced significant apoptosis in AML and B lymphoma cells by arresting the cell cycle at the G1/G0 or G2/M stage and blocked BTK autophosphorylation as well as the ensuing abrogation of pro-survival AKT and ERK signaling. Taken together, these results suggest that 11a and 11b might serve as valuable preclinical candidates for the treatment of AML and B-cell lymphoma.

Casein kinase 1α inhibits p53 downstream of MDM2‑mediated autophagy and apoptosis in acute myeloid leukemia.

Enhancement of autophagy serves as a promising therapeutic strategy for cancer, including acute myeloid leukemia (AML). Casein kinase 1α (CK1α), encoded by CSNK1A1, regulates Wnt/β-catenin, p53 and other key signaling pathways, and is critically involved in tumor progression. However, the relationship and mechanism of CK1α with autophagy in AML still remain unclear. In the present study, it was found that AML patients had higher expression of CSNK1A1 mRNA than healthy donors. Furthermore, we analyzed 163 cases of AML patients in the LAML database of TCGA and found that AML patients with high CSNK1A1 had shorter overall survival than those with low or medium CSNK1A1 expression. Furthermore, we demonstrated that CK1α was a negative regulator of autophagy and apoptosis. Pharmacologic inhibition of CK1α using D4476 or CK1α knockdown via lentivirus-mediated shRNA suppressed proliferation and the clone formation by enhancing autophagic flux and apoptosis in AML cell lines as well as in patient blast cells. Intriguingly, D4476-induced cell death was aggravated in combination with an autophagy inhibitor, Spautin-1, suggesting that autophagy may be a pro-survival signaling. CK1α interacted with murine double minute 2 (MDM2) and p53, and CK1α inhibitor D4476 significantly upregulated p53 and phosphorylated 5′ AMP-activated protein kinase (AMPK), and substantially inhibited the phosphorylation of mammalian target of rapamycin (mTOR). Our findings indicate that CK1α promotes AML by suppressing p53 downstream of MDM2-mediated autophagy and apoptosis, suggesting that targeting CK1α provides a therapeutic opportunity to treat AML.

AML-201: Synthetic Lethality in Acute Myeloid Leukemia: A Focus on Dihydroorotate Dehydrogenase Inhibitors

Context The inhibition of Dihydroorotate Dehydrogenase (DHODH) was recently found to induce differentiation and apoptosis in acute myeloid leukemia (AML). Multiple clinical trials testing DHODH inhibitors are currently ongoing, but the clinical effectiveness of these compounds is expected to be lower than in vitro due to: i) the heterogeneity of the AML genetic landscape and the well-known ability of leukemic cells to undergo clonal escape when treated with single agents, and ii) the pyrimidine salvage pathway, which could mitigate the pyrimidine starvation induced by DHODH inhibitors. Objective To increase the apoptotic rate induced by DHODH inhibitors in AML cells by testing multiple associations both with classical chemotherapeutical agents and targeted compounds, leveraging the synthetic lethality concept. Design MEDS433, the DHODH inhibitor developed by our group, was tested on several AML cell lines (THP1, MV4-11, NB4, U937, OCI-AML3) and primary cells, alone and in combination with cytarabine, anthracyclines, and decitabine. Moreover, in order to block the pyrimidine salvage pathway, MEDS433 was also combined with dipyridamole, a potent inhibitor of hENT1 and hENT2 nucleotide/nucleoside transport channels. Results The association of MEDS433 with cytarabine, idarubicin, and decitabine resulted in an additive effect, with a significant increase of the apoptotic rate in all tested AML cell lines. Moreover, the combination of MEDS433 with dipyridamole resulted in a synergistic effect, with an apoptotic rate ranging between 69% and 95% after just 3 days of treatment at the lowest effective MEDS433 concentration (0.1 μM), depending on the cell line. The combination of MEDS433, dipyridamole, and chemotherapy resulted in the massive death of all AML cells (>90%). The best associations were evaluated on primary cells, confirming optimal results. Conclusions The addition of DHODH inhibitors to the (3+7) regimen or to hypomethylating agents could significantly increase the performances of these classical treatments. Moreover, a deep pyrimidine starvation obtained through the inhibition of both DHODH and the pyrimidine salvage pathway, could further increase the antileukemic activity. Ongoing in vivo experiments will be determinant to verify this hypothesis and to assess the toxicity on normal cells. This work was supported by AIRC (IG-23344).

Choreography of methylation: KDM4C histone demethylation enables ALKBH5 m6A RNA demethylation to sustain AML stem cells.

Choreography of methylation: KDM4C histone demethylation enables ALKBH5 m6A RNA demethylation to sustain AML stem cells.

Cyclic AMP-hydrolyzing phosphodiesterase inhibitors potentiate statin-induced cancer cell death.

Dipyridamole, an antiplatelet drug, has been shown to synergize with statins to induce cancer cell-specific apoptosis. However, given the polypharmacology of dipyridamole, the mechanism by which it potentiates statin-induced apoptosis remains unclear. Here, we applied a pharmacological approach to identify the activity of dipyridamole specific to its synergistic anticancer interaction with statins. We evaluated compounds that phenocopy the individual activities of dipyridamole and assessed whether they could potentiate statin-induced cell death. Notably, we identified that a phosphodiesterase (PDE) inhibitor, cilostazol, and other compounds that increase intracellular cyclic adenosine monophosphate (cAMP) levels potentiate statin-induced apoptosis in acute myeloid leukemia and multiple myeloma cells. Additionally, we demonstrated that both dipyridamole and cilostazol further inhibit statin-induced activation of sterol regulatory element-binding protein 2, a known modulator of statin sensitivity, in a cAMP-independent manner. Taken together, our data support that PDE inhibitors such as dipyridamole and cilostazol can potentiate statin-induced apoptosis via a dual mechanism. Given that several PDE inhibitors are clinically approved for various indications, they are immediately available for testing in combination with statins for the treatment of hematological malignancies.

Tanshinone IIA regulates human AML cell proliferation, cell cycle, and apoptosis through miR-497-5p/AKT3 axis

BackgroundThe roots of Salvia miltiorrhiza are used in traditional Chinese medicine (TCM) and have high medicinal value. Tanshinone IIA (Tan IIA) is the active ingredient of Salvia miltiorrhiza which can inhibit the growth of acute leukemia cell lines in vitro, although the mechanism remains unclear.MethodsCCK-8 assays and BrdU stain were used to evaluate cell proliferation ability. Western blot analysis was used to detect protein expression. miR-497-5p expression level was detected by using qRT-PCR, and Annexin V-FITC/propidium iodide (PI) was used to detect cell apoptosis.ResultsHere we reported that Tan IIA could inhibit cell proliferation, induce cell cycle arrest, and promote cell apoptosis in acute myeloid leukemia (AML) cells. Thus, Tan IIA had the anti-cancer activity in AML cell lines, which was likely mediated by up-regulation of miR-497-5p expression. Our data further showed that in AML cells, the same effects were observed with overexpression of miR-497-5p by a miR-497-5p mimic. We demonstrated that Tan IIA could inhibit the expression of AKT3 by up-regulating the expression of miR-497-5p. We subsequently identified that AKT3 was the direct target of miR-497-5p, and that treatment with Tan IIA obviously reversed the effect of treatment with an miR-497-5p inhibitor under harsh conditions. In turn, PCNA expression was increased and cleaved Caspase-3 was suppressed, which contributed to the growth of AML cells.ConclusionsOur results showed that Tan IIA could inhibit cell proliferation in AML cells through miR-497-5p-mediated AKT3 downregulation pathway.

Knockdown of TRIM24 suppresses growth and induces apoptosis in acute myeloid leukemia through downregulation of Wnt/GSK-3β/β-catenin signaling.

Tripartite motif-containing protein 24 (TRIM24) has currently emerged as a crucial cancer-related gene present in a wide range of human cancer types. However, the involvement of TRIM24 in acute myeloid leukemia (AML) has not been well investigated. The present study aims to investigate the significance, cellular function, and potential regulatory mechanism of TRIM24 in AML. We found that TRIM24 expression was significantly upregulated in AML compared with normal tissues. AML patients with low expression of TRIM24 had higher survival rates than those expressing TRIM24 at higher levels. High expression of TRIM24 was also detected in AML cells and its knockdown markedly restricted proliferation and promoted apoptosis in AML cells. Further investigation revealed that TRIM24 contributed to the regulation of Wnt/β-catenin signaling, which was associated with modulating the phosphorylation status of glycogen synthase kinase-3β (GSK-3β). Inactivation of GSK-3β partially reversed the TRIM24 knockdown-mediated antitumor effects observed in AML cells. Furthermore, knockdown of TRIM24 retarded the growth of AML-derived xenograft tumors in nude mice in vivo. Overall, these findings demonstrate that knockdown of TRIM24 impedes the AML tumor growth through the modulation of Wnt/GSK-3β/β-catenin signaling. These findings highlight the potential TRIM24 as an attractive anticancer target to treat AML.

Effect of High Dose Vitamin C on Proliferation and Apoptosis of Acute Myeloid Leukemia Cells

To investigate the effects of high dose vitamin C on proliferation and apoptosis of acute myeloid leukemia (AML) cell lines including HL-60, U937 and primary CD34+ leukemia cells in AML. CD34+ cells were sorted by using immunomagnetic cell sorting system, then the primary CD34+ leukemia cells, including HL-60 and U937 cell lines were cultured in vitro. Cells in each group were treated with different concentrations of vitamin C, the survival rate of cells was determined by MTT assay, the apoptosis rate of cells was evaluated by Annexin V/PI double staining, the expression of apoptotic proteins-including cleaved caspase 3, cleaved caspase-9 and cleaved PARP were detected by Western blot. The proliferation of HL-60 and U937 cells could be inhibited by high dose vitamin C, which showed a concentration-dependent manner (r=-0.9664; r=-0.9796). HL-60 and U937 cells were treated with different concentrations of vitamin C (8 and 20 mmol/L) for 24 hours, respectively, it was found that with the increasing of vitamin C concentration, cell apoptosis rate was significantly increased (r=0.9905; r=0.9971), and the expression of apoptosis related proteins including cleaved caspase 3, cleaved caspase-9 and cleaved PARP was aslo significantly increased with the increasing of concentration. In addition, it was found that with or without the mutation of TET2, high dose vitamin C could inhibit the proliferation (r=-0.9719; r=-0.9699) and promote the apoptosis (r=0.9998; r=0.9901) of primary CD34+ leukemia cells in AML, which showed a dose-dependent manner, but it showed no effect on the proliferation (r=-0.2032) and apoptosis (r=0.1912) of normal CD34+ cells. High dose vitamin C can inhibit the proliferation and promote the apoptosis of acute myeloid leukemia cells, and selectively kill primary CD34+ leukemia cells in AML.

LncRNA SNHG5 regulates SOX4 expression through competitive binding to miR-489-3p in acute myeloid leukemia.

Currently, lncRNA plays an important role in the occurrence and development of acute myeloid leukemia (AML), including SNHG5. However, the role and mechanism of SNHG5 in AML remains unclear. In this study, we explored the regulatory mechanism of SNHG5 in the development of AML. QRT-PCR was used to investigate the expression of SNHG5, miR-489-3p, and SOX. The proliferation and apoptosis of AML cells were analyzed by cell transfection, cell counting kit-8 (CCK8), and flow cytometric analysis. Moreover, the expression analysis of marker proteins was detected by western blot. Through luciferase activity assay, RNA pull-down, and RNA-binding protein immunoprecipitation (RIP), we proved that SNHG5 could bind miR-489-3p and SOX4 which might be the target gene of miR-489-3p. We first found that SNHG5 was up-regulated in both AML patient bone marrow samples and various AML cell lines. Second, we found that knockdown of SNHG5 inhibited proliferation of AML cells and promoted apoptosis. It was found that SNHG5 could bind miR-489-3p, and the relative expression of SNHG5 was negatively correlated with miR-489-3p. Further results suggested that SOX4 might be the target gene of miR-489-3p. Finally, our experimental data indicated that knockdown of SNHG5 could reduce the tumor volume and down-regulated SOX4 levels in vivo. Our results demonstrated that SNHG5 affected the expression of SOX4 through binding miR-489-3p to regulate proliferation and apoptosis of AML, which might act as a prospective prognostic biological marker and a promising therapeutic target for AML.