Apoptosis In Leukemia Cells Research Articles

Lycorine induces apoptosis of acute myeloid leukemia cells and inhibits triglyceride production via binding and targeting FABP5.

Acute myeloid leukemia (AML) is the most common hematopoietic malignancy with abnormal lipid metabolism. However, currently available information on the involvement of the alterations in lipid metabolism in AML development is limited. In this study, we demonstrate that FABP5 expression facilitates AML cell viability, protects AML cells from apoptosis, and maintains triglyceride production. Our bioinformatics analysis revealed that FABP5 expression was upregulated and correlated with unfavorable overall survival of AML patients. FABP5 expression may be used to distinguish normal and AML with high accuracy. FABP5-based risk score was an independent risk factor for AML patients. AML patients with highly expressed FABP5 predicted resistance to drugs. In vitro study showed that FABP5 expression was remarkably elevated in primary AML blasts and an AML cell line. Silencing FABP5 expression attenuated AML cell viability, reduced triglyceride production and lipid droplet accumulation, and induced apoptosis. We utilized AutoDock online tool to identify lycorine as an FABP5 inhibitor by binding FABP5 at amino acid residues Ile54, Thr56, Thr63, and Arg109. Lycorine treatment downregulated the expression levels of FABP5 and its target PPARγ, impaired AML cell viability, triggered apoptosis, and reduced triglyceride production in AML cells. These results demonstrate that FABP5 is critical for AML cell survival and highlight a novel metabolic vulnerability for AML.

Cytarabine-induced destabilization of MCL1 mRNA and protein triggers apoptosis in leukemia cells

Although cytarabine (Ara-C) is the mainstay of treatment for acute myeloid leukemia (AML), its cytotoxic mechanisms for inducing apoptosis are poorly understood. Therefore, we investigated the Ara-C-induced cell death pathway in human AML U937 cells. Ara-C-induced downregulation of MCL1 is associated with the induction of mitochondrial depolarization and apoptosis. Ara-C triggered NOX4-mediated ROS production, which in turn activated p38 MAPK but inactivated AKT. Ara-C-induced DNA damage modulates p38 MAPK activation without affecting AKT inactivation in U937 cells. Inactivated AKT promotes GSK3β-dependent CREB phosphorylation, which in turn increases NOXA transcription, thereby triggering the degradation of MCL1 protein. Activated p38 MAPK induces HuR downregulation, leading to accelerated MCL1 mRNA turnover. A similar pathway also explains the Ara-C-induced THP-1 cell death. Collectively, our data confirm that Ara-C-triggered apoptosis in the AML cell lines U937 and THP-1 is mediated through the destabilization of MCL1 mRNA and protein. Furthermore, Ara-C acts synergistically with the BCL2 inhibitor ABT-199 to induce cell death in ABT-199-resistant and parental U937 cells by inhibiting MCL1 expression.

ACSM3 suppresses proliferation and induces apoptosis and cell cycle arrest in acute myeloid leukemia cells via the regulation of IGF2BP2.

Acyl-CoA medium-chain synthetase-3 (ACSM3) has been reported to be involved in the malignant progression of multiple types of human cancer. Nevertheless, the role of ACSM3 in acute myeloid leukemia (AML) and its exact mechanism of action are as yet undefined. In the present study, the expression levels of ACSM3 and IGF2 mRNA-binding protein 2 (IGF2BP2) were evaluated using the Gene Expression Profiling Interactive Analysis database and AML cells. The Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine staining were employed for the estimation of the cell proliferative activity. Induction of apoptosis and the assessment of the cell cycle were measured using flow cytometry and western blotting, respectively. The interaction of ACSM3 with IGF2BP2 was confirmed using an RNA immunoprecipitation assay. mRNA stabilization of ACSM3 following actinomycin D treatment was evaluated using reverse transcription-quantitative PCR analysis. The data indicated that the expression levels of ACSM3 were significantly downregulated, whereas those of IGF2BP2 were upregulated in tissues and AML cells. Downregulation of ACSM3 expression was closely associated with poor overall survival of patients with AML. ACSM3 overexpression repressed cell proliferative activity and induced apoptosis and cell cycle arrest. IGF2BP2 downregulated ACSM3 expression by reducing the stability of ACSM3 mRNA. In addition, IGF2BP2 overexpression counteracted the effects of ACSM3 overexpression noted on proliferation, induction of apoptosis and cell cycle arrest of HL-60 cells. In conclusion, ACSM3 repressed the cell proliferative activity and facilitated induction of apoptosis and cell cycle arrest in AML cells by modulating the expression of IGF2BP2.

Roles of the hsa_circ_0013880/USP32/Rap1b axis in the proliferation and apoptosis of acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a myeloid malignancy with generally high mortality. Although recent advances in AML research have revealed that circRNAs play significant roles in AML progression, our understanding of the leukemogenic mechanism of circRNAs remains very limited. In this study, increased expression of hsa_circ_0013880 was observed in bone marrow mononuclear cells (BMNCs) of AML patients. Overexpression of hsa_circ_0013880 promotes AML cell proliferation and migration and reduces cell apoptosis. Mechanistically, hsa_circ_0013880 could elevate the expression of USP32, a deubiquitinating enzyme that is highly expressed in the BMNCs of AML patients. Given the deubiquitination function of USP32, we further hypothesize that USP32 may mediate the malignant behaviors of AML cells by regulating the stability of Ras-related protein (Rap1b). At the molecular level, we find that silencing of USP32 increases ubiquitinated Rap1b. Overexpression of Rap1b restores the effects of USP32 knockdown, which further verifies our hypothesis. In addition, we propose another hypothesis that a potential regulatory network among hsa_circ_0013880, miR-148a-3p/miR-20a-5p and USP32 might exist in the development of AML, according to bioinformatics website predictions and our preliminary experimental verification. Overall, our findings will enrich the understanding of the hsa_circ_0013880/USP32/Rap1b axis in AML development, which may contribute to the development of novel therapeutic strategies for AML.

GIPC2 is a tumor suppressor gene for acute myeloid leukemia and induces apoptosis of leukemia cells by regulating the PI3K/AKT pathway.

Aims: To explore the expression and methylation levels of GIPC2 in acute myeloid leukemia (AML), discuss the mechanism of GIPC2 in AML and provide new strategies for the diagnosis and treatment of AML. Methods: qPCR, western blotting, cell counting kit-8 assay, bisulfite sequencing andother experiments were used in this study. Results: The expression of GIPC2 was found to be downregulated in AML and is mainly affected by DNA promoter methylation. Decitabine can demethylate the promoter region of GIPC2, and GIPC2 expression is upregulated after demethylation. Overexpression of GIPC2 in HL-60 cells can induce apoptosis by inhibiting the PI3K/AKT pathway. Conclusion: Our findings identify that GIPC2 is associated with the PI3K/AKT signaling pathway and may represent a potential therapeutic target and biomarker for the management of AML.

In vitro differentiation of myeloid suppressor cells (MDSC-like) from an immature myelomonocytic precursor THP-1

BackgroundMyeloid-derived suppressor cells (MDSCs) are a heterogeneous population with a potent suppressor profile that regulates immune responses. These cells are one of the main components of the microenvironment of several diseases, including solid and hematologic tumors, autoimmunities, and chronic inflammation. However, their wide use in studies is limited due to they comprehend a rare population, which is difficult to isolate, expand, differentiate, and maintain in culture. Additionally, this population has a complex phenotypic and functional characterization. ObjectiveTo develop a protocol for the in vitro production of MDSC-like population from the differentiation of the immature myeloid cell line THP-1. MethodsWe stimulated THP-1 with G-CSF (100 ng/mL) and IL-4 (20 ng/mL) for seven days to differentiate into the MDSC-like profile. At the end of the protocol, we characterized these cells phenotypically and functionally by immunophenotyping, gene expression analysis, cytokine release dosage, lymphocyte proliferation, and NK-mediated killing essays. ResultsWe differentiate THP-1 cells in an MDSC-like population, named THP1-MDSC-like, which presented immunophenotyping and gene expression profiles compatible with that described in the literature. Furthermore, we verified that this phenotypic and functional differentiation did not deviate to a macrophage profile of M1 or M2. These THP1-MDSC-like cells secreted several immunoregulatory cytokines into the microenvironment, consistent with the suppressor profile related to MDSC. In addition, the supernatant of these cells decreased the proliferation of activated lymphocytes and impaired the apoptosis of leukemic cells induced by NK cells. ConclusionsWe developed an effective protocol for MDSC in vitro production from the differentiation of the immature myeloid cell line THP-1 induced by G-CSF and IL-4. Furthermore, we demonstrated that THP1-MDSC-like suppressor cells contribute to the immune escape of AML cells. Potentially, these THP1-MDSC-like cells can be applied on a large-scale platform, thus being able to impact the course of several studies and models such as cancer, immunodeficiencies, autoimmunity, and chronic inflammation.

Exploring the mechanism of physcion-1-O-β-D-monoglucoside against acute lymphoblastic leukaemia based on network pharmacology and experimental validation

ObjectiveTo explore the mechanism of PG against acute lymphoblastic leukaemia (ALL) by network pharmacology and experimental verification in vitro. MethodsFirst, the biological activity of PG against B-ALL was determined by CCK-8 and flow cytometry. Then, the potential targets of PG were obtained from the PharmMapper database. ALL-related genes were collected from the GeneCards, OMIM and PharmGkb databases. The two datasets were intersected to obtain the target genes of PG in ALL. Then, protein interaction networks were constructed using the STRING database. The key targets were obtained by topological analysis of the network with Cytoscape 3.8.0 software. In addition, the mechanism of PG in ALL was confirmed by protein‒protein interaction, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. Furthermore, molecular docking was carried out by AutoDock Vina. Finally, Western blotting was performed to confirm the effect of PG on NALM6 cells. ResultsPG inhibited the proliferation of NALM6 cells. A total of 174 antileukaemic targets of PG were obtained by network pharmacology. The key targets included AKT1, MAPK14, EGFR, ESR1, LCK, PTPN11, RHOA, IGF1, MDM2, HSP90AA1, HRAS, SRC and JAK2. Enrichment analysis found that PG had antileukaemic effects by regulating key targets such as MAPK signalling, and PG had good binding activity with MAPK14 protein (−8.9 kcal/mol). PG could upregulate the expression of the target protein p-P38, induce cell cycle arrest, and promote the apoptosis of leukaemia cells. ConclusionMAPK14 was confirmed to be one of the key targets and pathways of PG by network pharmacology and molecular experiments.

Requirement for ER-mitochondria Ca2+ transfer, ROS production and mPTP formation in L-asparaginase-induced apoptosis of acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (aLL) is a malignant cancer in the blood and bone marrow characterized by rapid expansion of lymphoblasts. It is a common pediatric cancer and the principal basis of cancer death in children. Previously, we reported that L-asparaginase, a key component of acute lymphoblastic leukemia chemotherapy, causes IP3R-mediated ER Ca2+ release, which contributes to a fatal rise in [Ca2+]cyt, eliciting aLL cell apoptosis via upregulation of the Ca2+-regulated caspase pathway (Blood, 133, 2222-2232). However, the cellular events leading to the rise in [Ca2+]cyt following L-asparaginase-induced ER Ca2+ release remain obscure. Here, we show that in acute lymphoblastic leukemia cells, L-asparaginase causes mitochondrial permeability transition pore (mPTP) formation that is dependent on IP3R-mediated ER Ca2+ release. This is substantiated by the lack of L-asparaginase-induced ER Ca2+ release and loss of mitochondrial permeability transition pore formation in cells depleted of HAP1, a key component of the functional IP3R/HAP1/Htt ER Ca2+ channel. L-asparaginase induces ER Ca2+ transfer into mitochondria, which evokes an increase in reactive oxygen species (ROS) level. L-asparaginase-induced rise in mitochondrial Ca2+ and reactive oxygen species production cause mitochondrial permeability transition pore formation that then leads to an increase in [Ca2+]cyt. Such rise in [Ca2+]cyt is inhibited by Ruthenium red (RuR), an inhibitor of the mitochondrial calcium uniporter (MCU) that is required for mitochondrial Ca2+ uptake, and cyclosporine A (CsA), an mitochondrial permeability transition pore inhibitor. Blocking ER-mitochondria Ca2+ transfer, mitochondrial ROS production, and/or mitochondrial permeability transition pore formation inhibit L-asparaginase-induced apoptosis. Taken together, these findings fill in the gaps in our understanding of the Ca2+-mediated mechanisms behind L-asparaginase-induced apoptosis in acute lymphoblastic leukemia cells.

Polo-like kinase 1 Decrease During Induction Therapy Could Indicate Good Treatment Response, Favorable Risk Stratification, and Prolonged Survival in Pediatric Acute Lymphoblastic Leukemia.

Polo-like kinase 1 (PLK1) modulates leukemia cell apoptosis, proliferation, and cell cycle arrest in the progression of acute lymphoblastic leukemia (ALL). This study intended to investigate the dysregulation of PLK1 and its association with induction therapy response and prognosis in pediatric ALL patients. Bone marrow mononuclear cell samples were collected from 90 pediatric ALL patients at baseline and on the 15th day of induction therapy (D15), as well as from 20 controls after enrollment, for the detection of PLK1 by reverse transcription-quantitative polymerase chain reaction. PLK1 was increased in pediatric ALL patients compared with controls ( P <0.001). In pediatric ALL patients, PLK1 decreased from baseline to D15 ( P <0.001). Lower PLK1 at baseline was associated with a good prednisone response ( P =0.002), while decreased PLK1 at D15 was related to good prednisone response ( P =0.001), better bone marrow response ( P =0.025), and favorable risk stratification ( P =0.014). In addition, reduced PLK1 at baseline was linked with better event-free survival (EFS) ( P =0.046), and decreased PLK1 at D15 was related to prolonged EFS ( P =0.027) and overall survival (OS) ( P =0.047). Moreover, PLK1 decline ≥25% was linked to favorable EFS ( P =0.015) and OS ( P =0.008). Further multivariate Cox proportional regression analysis revealed that PLK1 decline ≥25% was independently linked with prolonged EFS (hazard ratio (HR)=0.324, P =0.024) and OS (HR=0.211, P =0.019). The reduction of PLK1 after induction therapy reflects a good treatment response and correlates with a favorable survival profile in pediatric ALL patients.

Calcium influx, oxidative stress, and apoptosis induced by TRPV1 in chronic myeloid leukemia cells: Synergistic effects with imatinib.

Introduction: Calcium flux is the master second messenger that influences the proliferation-apoptosis balance. The ability of calcium flux alterations to reduce cell growth makes ion channels interesting targets for therapy. Among all, we focused on transient receptor potential vanilloid 1, a ligand-gated cation channel with selectivity for calcium. Its involvement in hematological malignancies is poorly investigated, especially in the field of chronic myeloid leukemia, a malignancy characterized by the accumulation of immature cells. Methods: FACS analysis, Western blot analysis, gene silencing, and cell viability assay were performed to investigate the activation of transient receptor potential vanilloid 1, by N-oleoyl-dopamine, in chronic myeloid leukemia cell lines. Results: We demonstrated that the triggering of transient receptor potential vanilloid 1 inhibits cell growth and promotes apoptosis of chronic myeloid leukemia cells. Its activation induced calcium influx, oxidative stress, ER stress, mitochondria dysfunction, and caspase activation. Interestingly, a synergistic effect exerted by N-oleoyl-dopamine and the standard drug imatinib was found. Conclusion: Overall, our results support that transient receptor potential vanilloid 1 activation could be a promising strategy to enhance conventional therapy and improve the management of chronic myeloid leukemia.

Ceefourin-1, a MRP4/ABCC4 inhibitor, induces apoptosis in AML cells enhanced by histamine

BackgroundCeefourin-1 is a specific MRP4/ABCC4 inhibitor with potential antileukemic activity. In this study, we evaluate the ability of ceefourin-1 alone or in combination with histamine, an approved antileukemic agent, to induce cell differentiation or apoptosis in human acute myeloid leukemic cells. We also examine ceefourin-1 toxicity in mice. MethodsU937, HL-60, and KG1a cells were used as models for human acute myeloid leukemia. Cyclic AMP efflux was estimated by measuring intracellular and extracellular cAMP levels. Cell differentiation was assessed by levels of CD14 and CD11b by FACS, and CD88 by western blot, and by cell morphology. Apoptosis was evaluated by cleavage of caspase-3 and PARP by western blot, and by annexin V binding assay. Subacute toxicity study of ceefourin-1 was carried out in BALB/c mice. ResultsCeefourin-1 inhibits cAMP exclusion in AML cells and promotes intracellular signaling via CREB. Ceefourin-1 leads AML cells to apoptosis and histamine potentiates this effect, without evidence of cell differentiation. Intraperitoneal administration of ceefourin-1 shows no important alterations in mice blood parameters, hepatic, and renal functions, nor signs of histologic damage. ConclusionsThese results show that ceefourin-1 promotes apoptosis in AML cells that is enhanced by histamine.General significance:This work indicates that ceefourin-1 represents a promising molecule that could be used alone or in combination with histamine for in vivo evaluation in acute myeloid leukemia malignancies.

High expression level of the FTH1 gene is associated with poor prognosis in children with non-M3 acute myeloid leukemia.

Acute myelogenous leukemia (AML) is a disease that severely affects the physical health of children. Thus, we aimed to identify biomarkers associated with AML prognosis in children. Using transcriptomics on an mRNA dataset from 27 children with non-M3 AML, we selected genes from among those with the top 5000 median absolute deviation (MAD) values for subsequent analysis which showed that two modules were associated with AML risk groups. Thus, enrichment analysis was performed using genes from these modules. A one-way Cox analysis was performed on a dataset of 149 non-M3 AML patients downloaded from the TCGA. This identified four genes as significant: FTH1, RCC2, ABHD17B, and IRAK1. Through survival analysis, FTH1 was identified as a key gene associated with AML prognosis. We verified the proliferative and regulatory effects of ferroptosis on MOLM-13 and THP-1 cells using Liproxstatin-1 and Erastin respectively by CCK-8 and flow cytometry assays. Furthermore, we assayed expression levels of FTH1 in MOLM-13 and THP-1 cells after induction and inhibition of ferroptosis by real-time quantitative PCR, which showed that upregulated FTH1 expression promoted proliferation and inhibited apoptosis in leukemia cells. In conclusion, high expression of FTH1 promoted proliferation and inhibited apoptosis of leukemic cells through the ferroptosis pathway and is thus a potential risk factor that affects the prognosis of non-M3 AML in children.

Inhibitory Effect of Small Molecule Compound TIC10 on Leukemia

To investigate the inhibitory effect of small molecule compound TIC10 on multiple leukemia cells and its intervention effect on acute myeloid leukemia(AML) induced by MLL-AF9. Leukemia cells (MOLM-13, MV4-11, THP-1, Kasumi-1, KG-1 and K562) were used to detect the inhibitory effect of TIC10 on proliferation of leukemia cells through CCK-8 and cell count. The effect of TIC10 on the cell-cycle of leukemia cells was detected by flow cytometry with BrdU and DAPI staining. Apoptosis was detected by Annexin V and PI staining. Western blot was used to detect the effect of TIC10 on the expression of caspase3, a key apoptosis-associated protein. The intervention effect of TIC10 on the progression of leukemia in vivo was detected by constructing a mouse MLL-AF9 leukemia model. TIC10 treatment significantly inhibited the proliferation of MOLM-13, MV4-11, THP-1, and Kasumi-1 cells(P<0.05). TIC10 treatment substantially reduced the incorporation of BrdU in MV4-11 and Kasumi-1 cells (P<0.01), suggesting that TIC10 inhibited S-phase entry of leukemia cells. Apoptosis assay showed that TIC10 obviously induced apoptosis of leukemia cells(P<0.01). Western blot results showed that TIC10 treatment induced an elevated cleaved-caspase3 protein level in leukemia cells(P<0.05). The results of animal experiments proved that TIC10 effectively interfered with the progression of AML in vivo . TIC10 can effectively inhibite the proliferation of leukemia cells and induce apoptosis, and have a certain intervention effect on AML induced by MLL-AF9, indicating that TIC10 as a potential candidate drug for the treatment of leukemia.

Therapeutic implication of Pleurotus ostreatus on microRNAs expression in acute myeloid leukemia: A systematic review

Until now, the modality of choice for patients with acute myeloid leukemia (AML) is chemotherapy. However, chemotherapy has resulted in severe side effects, especially for AML patients aged over 60 years. In addition, cases of multidrug resistance (MDR) have been reported. Pleurotus ostreatus or oyster mushroom has been widely known to possess anticancer effects. The effect might be elicited by its active ingredients. On the other hand, miRNA as a therapeutic target is thought to interact with the active compounds of Pleurotus ostreatus, thus making it a potential complementary treatment for AML. This review was conducted to determine the effect of the active ingredients in Pleurotus ostreatus through their mechanism of action on certain miRNAs. The review was conducted using 49 literatures that have passed the quality assessment using the CRAAP criteria and the JBI checklist. The literature search was carried out using 4 databases, including PubMed, Research Gate, ProQuest, and Science Direct. The systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The results of the systematic review were synthesized in a narrative review without using statistical tests. In summary, at the molecular level, the active compounds in Pleurotus ostreatus can regulate several miRNAs that affect the proliferation and apoptosis of leukemia cells. In the future, it is necessary to carry out molecular and clinical analysis regarding the relationship between the active compounds of Pleurotus ostreatus and the expression of AML-related miRNAs.

DRP1 Inhibition Enhances Venetoclax-Induced Mitochondrial Apoptosis in TP53-Mutated Acute Myeloid Leukemia Cells through BAX/BAK Activation.

Although TP53 mutations in acute myeloid leukemia (AML) are associated with poor response to venetoclax, the underlying resistance mechanism remains unclear. Herein, we investigated the functional role of dynamin-related protein 1 (DRP1) in venetoclax sensitivity in AML cells with respect to TP53 mutation status. Effects of DRP1 inhibition on venetoclax-induced cell death were compared in TP53-mutated (THP-1 and Kasumi-1) and TP53 wild-type leukemia cell lines (MOLM-13 and MV4-11), as well as in primary AML cells obtained from patients. Venetoclax induced apoptosis in TP53 wild-type AML cells but had limited effects in TP53-mutated AML cells. DRP1 expression was downregulated in MOLM-13 cells after venetoclax treatment but was unaffected in THP-1 cells. Cotreatment of THP-1 cells with venetoclax and a TP53 activator NSC59984 downregulated DRP1 expression and increased apoptosis. Combination treatment with the DRP1 inhibitor Mdivi-1 and venetoclax significantly increased mitochondria-mediated apoptosis in TP53-mutated AML cells. The combination of Mdivi-1 and venetoclax resulted in noticeable downregulation of MCL-1 and BCL-xL, accompanied by the upregulation of NOXA, PUMA, BAK, and BAX. These findings suggest that DRP1 is functionally associated with venetoclax sensitivity in TP53-mutated AML cells. Targeting DRP1 may represent an effective therapeutic strategy for overcoming venetoclax resistance in TP53-mutated AML.

A novel Mcl-1 inhibitor synergizes with venetoclax to induce apoptosis in cancer cells

BackgroundEvading apoptosis by overexpression of anti-apoptotic Bcl-2 family proteins is a hallmark of cancer cells and the Bcl-2 selective inhibitor venetoclax is widely used in the treatment of hematologic malignancies. Mcl-1, another anti-apoptotic Bcl-2 family member, is recognized as the primary cause of resistance to venetoclax treatment. However, there is currently no Mcl-1 inhibitor approved for clinical use.MethodsPaired parental and Mcl-1 knockout H1299 cells were used to screen and identify a small molecule named MI-238. Immunoprecipitation (IP) and flow cytometry assay were performed to analyze the activation of pro-apoptotic protein Bak. Annexin V staining and western blot analysis of cleaved caspase 3 were employed to measure the cell apoptosis. Mouse xenograft AML model using luciferase-expressing Molm13 cells was employed to evaluate in vivo therapeutic efficacy. Bone marrow samples from newly diagnosed AML patients were collected to evaluate the therapeutic potency.ResultsHere, we show that MI-238, a novel and specific Mcl-1 inhibitor, can disrupt the association of Mcl-1 with BH3-only pro-apoptotic proteins, selectively leading to apoptosis in Mcl-1 proficient cells. Moreover, MI-238 treatment also potently induces apoptosis in acute myeloid leukemia (AML) cells. Notably, the combined treatment of MI-238 with venetoclax exhibited strong synergistic anti-cancer effects in AML cells in vitro, MOLM-13 xenografts mouse model and AML patient samples.ConclusionsThis study identified a novel and selective Mcl-1 inhibitor MI-238 and demonstrated that the development of MI-238 provides a novel strategy to improve the outcome of venetoclax therapy in AML.

CDK7 inhibition induces apoptosis in acute myeloid leukemia cells and exerts synergistic antileukemic effects with azacitidine in vitro and in vivo

THZ1, a CDK7 inhibitor, has potent antitumor effects in several cancers; however, its role in Acute myeloid leukemia (AML) is unclear. We explored the effects and potential mechanisms of THZ1, alone and in combination with azacitidine (AZA), in AML cells and xenograft models. THZ1 decreased cell viability, induced apoptosis in a dose and time-dependent manner, induced G0/G1 cell cycle arrest, decreased phosphorylated CDK1 and CDK2 expression, and inhibited RNA Pol II phosphorylation at multiple serine sites. The combination of AZA and THZ1 exhibited synergistic antileukemic effects in AML cell lines and primary cells with MCL1 and c-MYC downregulation. Moreover, the combination therapy significantly decreased tumor burden and prolonged animal survival in xenograft mice models. Our data demonstrate that CDK7 inhibition induces the apoptosis of AML cells and exerts a synergistic antileukemia effect with AZA in vitro and in vivo, which supports future exploration of this combination in clinical studies.

DNA hypermethylation-induced miR-182 silence targets BCL2 and HOXA9 to facilitate the self-renewal of leukemia stem cell, accelerate acute myeloid leukemia progression, and determine the sensitivity of BCL2 inhibitor venetoclax.

Rationale: microRNAs (miRNAs) are frequently deregulated and play important roles in the pathogenesis and progression of acute myeloid leukemia (AML). miR-182 functions as an onco-miRNA or tumor suppressor miRNA in the context of different cancers. However, whether miR-182 affects the self-renewal of leukemia stem cells (LSCs) and normal hematopoietic stem progenitor cells (HSPCs) is unknown. Methods: Bisulfite sequencing was used to analyze the methylation status at pri-miR-182 promoter. Lineage-negative HSPCs were isolated from miR-182 knockout (182KO) and wild-type (182WT) mice to construct MLL-AF9-transformed AML model. The effects of miR-182 depletion on the overall survival and function of LSC were analyzed in this mouse model in vivo. Results: miR-182-5p (miR-182) expression was lower in AML blasts than normal controls (NCs) with hypermethylation observed at putative pri-miR-182 promoter in AML blasts but unmethylation in NCs. Overexpression of miR-182 inhibited proliferation, reduced colony formation, and induced apoptosis in leukemic cells. In addition, depletion of miR-182 accelerated the development and shortened the overall survival (OS) in MLL-AF9-transformed murine AML through increasing LSC frequency and self-renewal ability. Consistently, overexpression of miR-182 attenuated AML development and extended the OS in the murine AML model. Most importantly, miR-182 was likely dispensable for normal hematopoiesis. Mechanistically, we identified BCL2 and HOXA9 as two key targets of miR-182 in this context. Most importantly, AML patients with miR-182 unmethylation had high expression of miR-182 followed by low protein expression of BCL2 and resistance to BCL2 inhibitor venetoclax (Ven) in vitro. Conclusions: Our results suggest that miR-182 is a potential therapeutic target for AML patients through attenuating the self-renewal of LSC but not HSPC. miR-182 promoter methylation could determine the sensitivity of Ven treatment and provide a potential biomarker for it.

MiR-125b-5p Suppresses Leukemia Cell Proliferation by Regulating MCL1

Leukemia threatens children's health, and leukemia cell proliferation and apoptosis participate in the regulation of leukemia. The current study aims to probe into the miR-125b-5p biological function in regulating leukemia cell proliferation and apoptosis by myeloid cell leukemia 1 (MCL1). Quantitative real-time polymerase chain reaction was conducted to quantify miR-125b-5p expression in leukemia cells. Cell transfection, cell-counting assay 8, Western blot, and flow cytometry assays were applied to assess the miR-125b-5p function in leukemia. A dual-luciferase reporter gene assay was applied to investigate the mechanism. miR-125b-5p was lessened in leukemia cells, and the increased miR-125b-5p repressed leukemia cell proliferation and boosted apoptosis. Further, miR-125b-5p could bound with the MCL1 3'-untranslated region and regulated its expression. Furthermore, the elevated expression of miR-125b-5p repressed leukemia cell proliferation and boosted apoptosis through downregulating MCL1. miR-125b-5p inhibited leukemia cell proliferation and boosted apoptosis through decreasing MCL1.

PD-1 AND TIM-3 BLOCKING CANNOT ENHANCE APOPTOSIS OF CHRONIC LYMPHOCYTIC LEUKEMIA CELLS INDUCED BY PERIPHERAL BLOOD CD8+ T CELLS.

Given theinvaluable success of immune checkpoint inhibitors for tumor immunotherapy, in this study, theeffect of programmed cell death 1 (PD-1)and T cell immunoglobulin-3 (TIM-3)blocking was investigated to induce apoptosis of leukemic cells by exhausted CD8+ T cells in patients with chronic lymphocytic leukemia (CLL). Peripheral blood CD8+ T cells were positively isolated from 16CLL patients using magnetic beads separation method. Isolated CD8+ T cells were treated with either blocking anti-PD-1, anti-TIM-3and isotype-matched control antibodies and then co-cultured with CLL leukemic cells as target. Thepercentage of apoptotic leukemic cells and theexpression of apoptosis-related genes were evaluated by flow cytometry and real-time polymerase chain reaction methods, respectively. Interferon gamma and tumor necrosis factor alpha concentration was also measured by ELISA. Flow cytometric analysis of apoptotic leukemic cells indicated that theblockade of PD-1and TIM-3did not significantly enhance theapoptosis of CLL cells by CD8+ T cells, which then were confirmed by analysis of BAX, BCL2and CASP3gene expression, which was similar in blocked and control groups. No significant difference was found between blocked and control groups in terms of interferon gamma and tumor necrosis factor alpha production by CD8+ T cells. We concluded that theblockade of PD-1and TIM-3is not an effective strategy to restore thefunction of CD8+ T cells in CLL patients at theearly clinical stages of thedisease. Further in vitro and in vivo studies are needed to more address theapplication of immune checkpoint blockade in CLL patients.