Apoptosis In Leukemia Cells Research Articles

Dihydroxyquingdainone Induces Apoptosis in Leukaemia and Lymphoma Cells via the Mitochondrial Pathway in a Bcl-2- and Caspase-3-Dependent Manner and Overcomes Resistance to Cytostatic Drugs In Vitro.

Isatis tinctoria and its indigo dyes have already provided highly active anti-leukaemic lead compounds, with the focus mainly being on indirubin, whereas indigo itself is inactive. There are many more indigoids to find in this plant extract, for example, quingdainone, an indigoid derived from tryptanthrin. We present here a new synthesis of hitherto neglected substituted quingdainones, which is very necessary due to their poor solubility behaviour, and a structure-dependent anti-leukaemic activity study of a number of compounds. Substituted α-phenylaminoacrylic acid was synthesised by hydrogen sulfide extrusion from an analogue mercaptoacetic acid, available from the condensation of rhodanin and a substituted tryptanthrin. It is shown that just improving water solubility does not increase anti-leukaemic activity, since a quingdainone carboxylic acid is inactive compared to dihydroxyquingdainone. The most effective compound, dihydroxyquingdainone with an AC50 of 7.5 µmole, is further characterised, revealing its ability to overcome multidrug resistance in leukaemia cells (Nalm-6/BeKa) with p-glycoprotein expression.

Small molecule MMRi62 targets MDM4 for degradation and induces leukemic cell apoptosis regardless of p53 status.

MDM2 and MDM4 proteins are key negative regulators of tumor suppressor p53. MDM2 and MDM4 interact via their RING domains and form a heterodimer polyubiquitin E3 ligase essential for p53 degradation. MDM4 also forms heterodimer E3 ligases with MDM2 isoforms that lack p53-binding domains, which regulate p53 and MDM4 stability. We are working to identify small-molecule inhibitors targeting the RING domain of MDM2-MDM4 (MMRi) that can inactivate the total oncogenic activity of MDM2-MDM4 heterodimers. Here, we describe the identification and characterization of MMRi62 as an MDM4-degrader and apoptosis inducer in leukemia cells. Biochemically, in our experiments, MMRi62 bound to preformed RING domain heterodimers altered the substrate preference toward MDM4 ubiquitination and promoted MDM2-dependent MDM4 degradation in cells. This MDM4-degrader activity of MMRi62 was found to be associated with potent apoptosis induction in leukemia cells. Interestingly, MMRi62 effectively induced apoptosis in p53 mutant, multidrug-resistant leukemia cells and patient samples in addition to p53 wild-type cells. In contrast, MMRi67 as a RING heterodimer disruptor and an enzymatic inhibitor of the MDM2-MDM4 E3 complex lacked MDM4-degrader activity and failed to induce apoptosis in these cells. In summary, this study identifies MMRi62 as a novel MDM2-MDM4-targeting agent and suggests that small molecules capable of promoting MDM4 degradation may be a viable new approach to killing leukemia cells bearing non-functional p53 by apoptosis.

Induction of zinc finger protein RNF6 auto-ubiquitination for the treatment of myeloma and chronic myeloid leukemia

The zinc finger ubiquitin ligase RNF6 has been proposed as a potential therapeutic target in several cancers, but understanding its molecular mechanism of degradation has been elusive. In the present study, we find that RNF6 is degraded via auto-ubiquitination in a manner dependent on its Really Interesting New Gene (RING) domain. We determine that when the RING domain is deleted (ΔRING) or the core cysteine residues in the zinc finger are mutated (C632S/C635S), the WT protein, but not the ΔRING or mutant RNF6 protein, undergoes polyubiquitination. We also identify USP7 as a deubiquitinase of RNF6 by tandem mass spectrometry. We show that USP7 interacts with RNF6 and abolishes its K48-linked polyubiquitination, thereby preventing its degradation. In contrast, we found a USP7-specific inhibitor promotes RNF6 polyubiquitination, degradation, and cell death. Furthermore, we demonstrate the anti-leukemic drug Nilotinib and anti-myeloma drug Panobinostat (LBH589) induce RNF6 K48-linked polyubiquitination and degradation in both multiple myeloma (MM) and leukemia cells. In agreement with our hypothesis on the mode of RNF6 degradation, we show these drugs promote RNF6 auto-ubiquitination in an in vitro ubiquitination system without other E3 ligases. Consistently, reexpression of RNF6 ablates drug-induced MM and leukemia cell apoptosis. Therefore, our results reveal that RNF6 is a RING E3 ligase that undergoes auto-ubiquitination, which could be abolished by USP7 and induced by anti-cancer drugs. We propose that chemical induction of RNF6 auto-ubiquitination and degradation could be a novel strategy for the treatment of hematological malignancies including MM and leukemia.

5-Demethylnobiletin Inhibits Cell Proliferation, Downregulates ID1 Expression, Modulates the NF-κB/TNF-α Pathway and Exerts Antileukemic Effects in AML Cells.

Acute myeloid leukemia (AML) is characterized by the dysregulation of hematopoietic cell proliferation, resulting in the accumulation of immature myeloid cells in bone marrow. 5-Demethylnobiletin (5-demethyl NOB), a citrus 5-hydroxylated polymethoxyflavone, has been reported to exhibit various bioactivities, such as antioxidant, anti-inflammatory and anticancer properties. In this study, we investigated the antileukemic effects of 5-demethyl NOB and its underlying molecular mechanisms in human AML cells. We found that 5-demethyl NOB (20–80 μM) significantly reduced human leukemia cell viability, and the following trend of effectiveness was observed: THP-1 ≈ U-937 > HEL > HL-60 > K562 cells. 5-Demethyl NOB (20 and 40 μM) modulated the cell cycle through the regulation of p21, cyclin E1 and cyclin A1 expression and induced S phase arrest. 5-Demethyl NOB also promoted leukemia cell apoptosis and differentiation. Microarray-based transcriptome, Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) of differentially expressed genes (DEGs) analysis showed that the expression of inhibitor of differentiation/DNA binding 1 (ID1), a gene associated with the GO biological process (BP) cell population proliferation (GO: 0008283), was most strongly suppressed by 5-demethyl NOB (40 μM) in THP-1 cells. We further demonstrated that 5-demethyl NOB-induced ID1 reduction was associated with the inhibition of leukemia cell growth. Moreover, DEGs involved in the hallmark gene set NF-κB/TNF-α signaling pathway were markedly enriched and downregulated by 5-demethyl NOB. Finally, we demonstrated that 5-demethyl NOB (20 and 40 μM), combined with cytarabine, synergistically reduced THP-1 and U-937 cell viability. Our current findings support that 5-demethyl NOB dramatically suppresses leukemia cell proliferation and may serve as a potential phytochemical for human AML chemotherapy.

Bioinformatics Analysis Reveals FOXM1/BUB1B Signaling Pathway as a Key Target of Neosetophomone B in Human Leukemic Cells: A Gene Network-Based Microarray Analysis.

Abnormal expression of Forkhead box protein M1 (FOXM1) and serine/threonine kinase Budding uninhibited by benzimidazoles 1 (BUB1B) contributes to the development and progression of several cancers, including chronic myelogenous leukemia (CML). However, the molecular mechanism of the FOXM1/BUB1B regulatory network and the role of Neosetophomone-B (NSP-B) in leukemia remains unclear. NSP-B, a meroterpenoid fungal secondary metabolite, possesses anticancer potential in human leukemic cells lines; however, the underlying mechanism has not been elucidated. The present study aimed to explore the role of NSP-B on FOXM1/BUB1B signaling and the underlying molecular mechanism of apoptosis induction in leukemic cells. We performed gene expression profiling of NSP-B-treated and untreated leukemic cells to search for differentially expressed genes (DEGs). Interestingly BUB1B was found to be significantly downregulated (logFC -2.60, adjusted p = 0.001) in the treated cell line with the highest connectivity score among cancer genes. Analysis of TCGA data revealed overexpression of BUB1B compared to normal in most cancers and overexpression was associated with poor prognosis. BUB1B also showed a highly significant positive correlation with FOXM1 in all the TCGA cancer types. We used human leukemic cell lines (K562 and U937) as an in vitro study model to validate our findings. We found that NSP-B treatment of leukemic cells suppressed the expression of FOXM1 and BUB1B in a dose-dependent manner. In addition, NSP-B also resulted in the downregulation of FOXM1-regulated genes such as Aurora kinase A, Aurora kinase B, CDK4, and CDK6. Suppression of FOXM1 either by siRNA or NSP-B reduced BUB1B expression and enhanced cell survival inhibition and induction of apoptosis. Interestingly combination treatment of thiostrepton and NSP-B suppressed of cell viability and inducted apoptosis in leukemic cells via enhancing the activation of caspase-3 and caspase-8 compared with single-agent treatment. These results demonstrate the important role of the FOXM1/BUB1B pathway in leukemia and thus a potential therapeutic target.

Ligustrum vulgare leaves and fruit extracts induce apoptosis of human leukemia cells

Background and purpose: Cytotoxic effects of Ligustrum vulgare leaves on HeLa cervical tumor cells suggested that Ligustrum vulgare extracts should be investigated as potential anticancer agents. Therefore, we examined a potential antileukemic activity of methanolic extracts of Ligustrum vulgare leaves and fruit extracts on two types of leukemia cells, MOLT-4 and JVM-13, lymphocytes isolated from the blood of 33 chronic lymphocytic leukemia (CLL) patients and on mononuclear leukocytes isolated from the blood of 18 healthy individuals.Material and methods: The cytotoxicity of examined extracts was measured by MTT assay and LDH activity test. The antiapoptotic potential of tested extracts was measured by Annexin V/7AAD flowcytometric assay.Results: The results showed that both extracts exhibited a moderate cytotoxic effect on all three types of leukemia cells. The Ligustrum vulgare leaf extract was the most effective on MOLT-4 cells, the fruit extract on JVM-13 cells and both extracts were equally effective on CLL cells. In addition, none of the tested extracts was toxic to healthy mononuclear cells. Both extracts acted by inducing apoptosis of leukemic cells.Conclusion: Ligustrum vulgare extracts exhibit significant antileukemic potential and should be further investigated.

Decitabine Enhances Acute Myeloid Leukemia Cell Apoptosis through SH3BGRL Upregulation.

SH3-domain-binding glutamic acid-rich protein-like protein (SH3BGRL) is downregulated in acute myeloid leukemia (AML). Clinically, DNA demethylating drug decitabine (DAC) combined with traditional chemotherapies reveals better efficacy on AML patients than the conventional chemotherapies alone. Our previous results revealed that human SH3-domain-binding glutamic acid-rich protein-like protein (SH3BGRL) plays a tumor suppressive role in AML but whether there is a connection between DAC and SH3BGRL expression remains elusive. Here, we tentatively treated AML cell lines U937, MV4, and HL-60 with DAC and Western Blots, RT-PCR was used to detect the expression of SH3BGRL. Cell proliferation and apoptosis were determined using Annexin V/7- AAD staining. Real-time RT-PCR and Western blot were used to determine the expression of SH3BGRL mRNA and protein. Methylation-specific PCR was used to quantify the DNA methylation in AML cell lines. DAC had cytotoxicity in HL-60, MV4, and U937. In U937 cell lines, treatment with DAC showed the upregulation of cleaved caspase3, PARP, and SH3BGRL. Upon treatment, up-regulation of SH3BGRL mRNA and protein was dose-dependent and this activity was partially inhibited in endogenous SH3BGRL knockdown cell lines. Thus, our results demonstrated a possibly cytotoxic role of DAC on AML cells by upregulation of SH3BGRL expression at epigenetic modulation level and the methylation status in the SH3BGRL promoter region could be a supplemental diagnostic marker to the precise administration of DAC to AML patients.

Investigating the potential therapeutic role of targeting STAT3 for overcoming drug resistance by regulating energy metabolism in chronic myeloid leukemia cells.

Objective(s):STATs are one of the initial targets of emerging anti-cancer agents due to their regulatory roles in survival, apoptosis, drug response, and cellular metabolism in CML. Aberrant STAT3 activity promotes malignancy, and acts as a metabolic switcher in cancer cell metabolism, contributing to resistance to TKI nilotinib. To investigate the possible therapeutic effects of targeting STAT3 to overcome nilotinib resistance by evaluating various cellular responses in both sensitive and nilotinib resistant CML cells and to test the hypothesis that energy metabolism modulation could be a mechanism for re-sensitization to nilotinib in resistant cells.Materials and Methods:By using RNAi-mediated STAT3 gene silencing, cell viability and proliferation assays, apoptotic analysis, expressional regulations of STAT mRNA transcripts, STAT3 total, pTyr705, pSer727 protein expression levels, and metabolic activity as energy metabolism was determined in CML model K562 cells, in vitro.Results:Targeting STAT3 sensitized both parental and especially nilotinib resistant cells by decreasing leukemic cell survival; inducing leukemic cell apoptosis, and decreasing STAT3 mRNA and protein expression levels. Besides, cell energy phenotype was modulated by switching energy metabolism from aerobic glycolysis to mitochondrial respiration in resistant cells. RNAi-mediated STAT3 silencing accelerated the sensitization of leukemia cells to nilotinib treatment, and STAT3-dependent energy metabolism regulation could be another underlying mechanism for regaining nilotinib response.Conclusion:Targeting STAT3 is an efficient strategy for improving the development of novel CML therapeutics for regaining nilotinib response, and re-sensitization of resistant cells could be mediated by induced apoptosis and regulation in energy metabolism.

P438: ARYL HYDROCARBON RECEPTOR SIGNALLING IN NORMAL HAEMATOPOIETIC (HSC) AND LEUKEMIA STEM CELLS (LSC): DIFFERENTIAL EFFECT OF AHR ANTAGONIST ON LSC COMPARED TO HSC

Background: Acute myeloid leukaemia (AML) is a devastating disease with poor prognosis. It is driven by leukemia stem cells (LSCs), which are able to survive treatment and drive relapse. This highlights the importance of characterizing LSCs. LSC-targeted treatment strategies are limited because there are no known surface markers that are specific for LSCs in every leukaemia. Targeting a signalling pathway that is altered in all LSCs could be a novel, promising treatment strategy. The aryl hydrocarbon receptor (AHR) pathway was shown to be critical for the self-renewal properties of normal haematopoietic stem cells (HSCs). Aims: We aimed to study the consequences of manipulating the AHR pathway in vivo in a murine leukaemia model. Methods: We used the following methods: murine bone marrow transplantation leukemia model (MBMTLM) - CALM-AF10 with limiting dilution assay (LDA); competitive repopulation assay (CRA) with LDA (CD45.2 donors into CD45.1 recipient mice), transcriptome analysis (sequencing with the Illumina platform and analisys with the DESeq2). Treatment of the cells was performed at various experiments with the AHR agonist, ITE, and an AHR antagonist, CH-223191 (a vehicle control is DMSO). Results: We used a murine retroviral transduction bone marrow transplantation leukemia model (MBMTLM)) with the CALM-AF10 minimal fusion gene (CAMF) as the oncogenic driver, to study the effect of AHR agonist (ITE) and an AHR antagonist (CH-223191) treatment on LSCs. Interestingly, both AHR agonist and antagonist treatment affected LSC frequency and survival of mice transplanted with treated cells. The LSC frequency as measured by limiting dilution transplantation assays (LDA) was 3.7 times lower after agonist treatment (1:163, blue survival curve in fig. 1, A) compared to a vehicle control (1:44, DMSO, black survival curve)). Very interestingly, after antagonist treatment the LSC frequency was so low that could not be measured as none of the mice transplanted with antagonist-treated cells in the LDAs developed leukaemia (n=9, CH-223191 (red curve in fig. 1, A)). Of note, our CAMF MBMTLM has a very high LSC frequency of about 1:4 in cells that were not cultured after thawing. We performed RNA-Seq on 90 samples to determine changes in gene expression patterns after treatment of healthy bone marrow cells and CAMF leukemia cells with the AHR agonist and antagonist. Preliminary differential expression analysis with DESeq2 revealed differential expression of genes involved in apoptosis and proliferation in leukaemia cells treated with CH-223191. As leukemia did not develop in recipients of cells treated with the antagonist CH-223191, genes differentially regulated in these cells might be critical and specific to LSC function and will be candidate targets for LSC-specific treatments. To confirm that AHR antagonist treatment affects LSCs differently than normal HCS, we performed competitive repopulation assay (CRA). Our preliminary data (n = 12) show that treatment of HSCs with CH-223191 results in a steady increase in the engraftment over a 47-week period following transplantation (coral bars, fig. 1, B), as opposed to the grafts from the ITE-treated arm (blue bars), vehicle or untreated HSCs (black and green bars, respectively). Image:Summary/Conclusion: In summary, our data show that the LSCs react differently than normal HSCs to changes in AHR signalling. These differences suggest it might be possible to specifically inhibit LSCs while sparing normal HSCs, thus paving the way to develop strategies to treat leukemia based on manipulating AHR signalling.

PB2207: IN VITRO VALIDATION OF DENDRIMER-BASED APPROACH FOR MICRO-RNA DELIVERY INTO LEUKEMIA CELLS

Background: microRNAs are known to be important players in the epigenetic regulation of many cell processes. Some of them demonstrate anti-tumor properties (e.g. miR-34), others are associated with the enhancing of the tumor growth (e.g. miR-21), therefore being prospective effectors and targets for the gene therapy. For this aim, synthetic microRNAs or microRNA inhibitors can be used. We suggest to use dendrimers - highly symmetric hyperbranched polymers - as a novel type of nanocarriers for therapeutic RNAs delivery and gene therapy. Aims: In this work we assessed the effects of microRNA-34a (miR-34a) and microRNA-21 inhibitor (anti-miR-21) on Jurkat and 1301 cells (human T-cell leukemia) upon the dendrimer-mediated delivery. Methods: As carriers for microRNAs, amphiphilic second- and third generation triazine-carbosilane dendrons (DG2 and DG3 respectively) and fully symmetrical 3rd generation carbosilane (BDEF33) and phosphorus (AE2G3) dendrimers have been taken. Oligonucleotides were complexed onto the surface of dendrimer vesicles by means of electrostatic interactions, forming dendriplexes. To prove the dendriplexes can be sufficiently internalized, we performed FACS analysis after 3 h of incubating cells with FITC-labelled microRNA. Cells were cultivated with free dendrimers or complexes (25, 50, 100, 150 nM RNA, 10-fold excess of cations) in the complete cell medium for 72 h. We investigated cell viability (MTT assay, Annexin V/PI apoptosis assay) in comparison with non-tumor cells (PBMCs), IL-10 secretion and expression of several markers, characterizing interaction of tumor with immune microenvironment - PD-L1, TIM-3, CD47. Statistical analysis was done by using Mann-Whitney and Wilcoxon tests, differences between groups were considered significant if p<0.05. This work was supported by RFBR grant No.18-33-20109, by the grant of the President of the Russian Federation No.2278.2019.4, by the HHU STIBET Scholarship grant support and based upon work from COST Action CA 17140 “Cancer Nanomedicine from the Bench to the Bedside” supported by COST (European Cooperation in Science and Technology). Results: Free dendrimers have their own dose-dependent toxic effects, which is slightly higher for tumor cells, than for PBMCs. Treatment by free dendritic molecules led to increasing of expression of PD-L1, TIM-3 and CD47 on Jurkat cells, but decreased the IL-10 secretion. Dendriplexes can be efficiently internalized into tumor cells, transport activity of dendrimers was higher than for Lipofectamine 3000 or was comparable. Dendron-based dendriplexes (DG2, DG3) had pro-apoptotic effect on leukemia cells. PD-L1 expression did not change after treatment by dendriplexes. Summary/Conclusion: Dendrimers not onle have their own toxic effect per se, but also they can be used as effective nanocarriers for therapeutic microRNAs. Using of complexes, containing microRNAs with anti-tumor activity, leads to induction of apoptosis of leukemia cells. Treatment by free dendrimers, but not by dendriplexes, increases expression of PD-L1, TIM-3, CD47 on Jurkat cells, but IL-10 secretion was found to be decreased. We suppose, that dendrimer-based approach can be useful and perspective tool for delivery of therapeuthic microRNAs into tumor cells. At the same moment, characterization of their effects on interactions between tumor and immune microenvironment demands the further studies.

MiRNA-222-3p enhances the proliferation and suppresses the apoptosis of acute myeloid leukemia cells by targeting Axin2 and modulating the Wnt/β-catenin pathway

MicroRNA (miRNA)-222-3p is overexpressed in numerous tumors, where it acts as an oncogene. Although miRNA-222 is highly expressed in acute myeloid leukemia (AML), its functions and the mechanisms underlying these functions have not yet been fully elucidated. This study aimed to investigate the regulatory roles of miRNA-222-3p in AML and the molecular mechanisms underlying these roles. In this study, we observed that miRNA-222-3p increased the viability and suppressed the apoptosis of AML cells. Axin2 was demonstrated to be a direct target of miRNA-222-3p, which when overexpressed, inhibited Axin2 expression and stimulated the Wnt/β-catenin pathway. In contrast, upregulation of Axin2 expression levels reduced the viability and enhanced the apoptosis of AML cells. Moreover, it partially reversed the effects of the miRNA-222-3p mimic on the proliferation and apoptosis of, and modulation of the Wnt/β-catenin pathway in, AML cells. Taken together, this study provides strong evidence that miRNA-222-3p can serve as a molecular target for AML treatment.

Inhibition of DEK Enhances Doxorubicin-Induced Apoptosis and Cell Cycle Arrest in T-Cell Acute Lymphoblastic Leukemia Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a serious hematological tumor derived from early T-cell progenitors, which is extremely resistant to chemotherapy. Classically, doxorubicin (DOX) is an effective first-line drug for the treatment of T-ALL; however, DOX resistance limits its clinical effect. The DEK proto-oncogene (DEK) has been involved in neoplasms but remains unexplored in T-ALL. We silenced DEK on Jurkat cells and detected cell proliferation with cell counting and colony formation assay. Then, we detected DEK's drug sensitivity to DOX with CCK-8, cell cycle, and apoptosis with DOX treatment. Western blot analysis was performed to determine protein expression of apoptosis and cell cycle-related genes, including BCL2L1, caspase-3, and cyclin-dependent kinases (CDK). Finally, the tumorigenic ability of DEK was analyzed using a BALB/C nude mouse model. In this study, DEK was highly expressed in Jurkat cells. Inhibition of DEK can lead to decreased cell proliferation and proportion of S-phase cells in the cell cycle and more cell apoptosis, and the effect is more obvious after DOX treatment. Western blot results showed that DOX treatment leads to cell cycle arrest, reduction of cyclin-dependent kinase 6 (CDK6) protein, accumulation of CDKN1A protein, and DOX-induced apoptosis accompanied by reductions in protein levels of BCL2L1, as well as increases in protein level of caspase-3. Furthermore, DEK-silenced Jurkat cells generated a significantly smaller tumor mass in mice. Our study found that DEK is a novel, potential therapeutic target for overcoming DOX resistance in T-ALL.

Abstract 3982: Molecular mechanism of cisplatin-induced p53 activation, cell cycle regulation, and apoptosis in acute leukemia cells

Abstract Cisplatin (CDDP) is an anticancer drug successfully used in the treatment of various human malignancies. Although it has been shown to enhance the potency of arsenic trioxide in chronic myelogenous leukemia (CML) cells and to induce cytotoxicity in acute promyelocytic leukemia (APL) cell lines, its molecular mechanism of action is mostly unknown. We investigated a novel mode of action of CDDP through modulation of stress signals, disruption of MDM2-DAXX-HAUSP complex, and activation of p53 in APL cell lines. We found that CDDP-induced stress signals were transmitted and reduced expression of complex molecules and also effected their association leading to p53 activation. CDDP-activated p53 led to cell cycle arrest and induced both early and late apoptosis in APL cells. Our findings also showed that CDDP stimulated more promyelocytes formation with dense granules, activated p53 expression, and reduced expression of MDM2 in both APL mice bone marrow cells and liver tissues. This novel mechanism of action of CDDP in APL cells may help in the design of new anti-leukemic drugs for treatment of APL patients. Citation Format: Paul B. Tchounwou, Sanjay Kumar. Molecular mechanism of cisplatin-induced p53 activation, cell cycle regulation, and apoptosis in acute leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3982.

Profiling the Effect of Targeting Wild Isocitrate Dehydrogenase 1 (IDH1) on the Cellular Metabolome of Leukemic Cells.

Leukemia is one of the most common primary malignancies of the hematologic system in both children and adults and remains a largely incurable or relapsing disease. The elucidation of disease subtypes based on mutational profiling has not improved clinical outcomes. IDH1/2 are critical enzymes of the TCA cycle that produces α-ketoglutarate (αKG). However, their mutated version is well reported in various cancer types, including leukemia, which produces D-2 hydroxyglutarate (D-2HG), an oncometabolite. Recently, some studies have shown that wild-type IDH1 is highly expressed in non-small cell lung carcinoma (NSCLC), primary glioblastomas (GBM), and several hematological malignancies and is correlated with disease progression. This work shows that the treatment of wild-type IDH1 leukemia cells with a specific IDH1 inhibitor shifted leukemic cells toward glycolysis from the oxidative phosphorylation (OXPHOS) phenotype. We also noticed a reduction in αKG in treated cells, possibly suggesting the inhibition of IDH1 enzymatic activity. Furthermore, we found that IDH1 inhibition reduced the metabolites related to one-carbon metabolism, which is essential for maintaining global methylation in leukemic cells. Finally, we observed that metabolic alteration in IDH1 inhibitor-treated leukemic cells promoted reactive oxygen species (ROS) formation and the loss of mitochondrial membrane potential, leading to apoptosis in leukemic cells. We showed that targeting wild-type IDH1 leukemic cells promotes metabolic alterations that can be exploited for combination therapies for a better outcome.

Hes1 Controls Proliferation and Apoptosis in Chronic Lymphoblastic Leukemia Cells by Modulating PTEN Expression.

Hairy and enhancer of split homolog-1 (HES1), regulated by the Notch, has been reported to play important roles in the immune response and cancers, such as leukemia. In this study, we aim to explore the effect of HES1-mediated Notch1 signaling pathway in chronic lymphocytic leukemia (CLL). Reverse transcription quantitative polymerase chain reaction and Western blot assay were conducted to determine the expression of HES1, Notch1, and PTEN in B lymphocytes of peripheral blood samples of 60 CLL patients. We used lentivirus-mediated overexpression or silencing of HES1 and the Notch1 signaling pathway inhibitor, MW167, to detect the interaction among HES1, Notch1, and PTEN in CLL MEC1 and HG3 cells. MTT assay and flow cytometry were employed for detection of biological behaviors of CLL cells. HES1 and Notch1 showed high expression, but PTEN displayed low expression in B lymphocytes of peripheral blood samples of patients with CLL in association with poor prognosis. HES1 bound to the promoter region of PTEN and reduced PTEN expression. Overexpression of HES1 activated the Notch1 signaling pathway, thus promoting the proliferation of CLL cells, increasing the proportion of cells arrested at the S phase and limiting the apoptosis of CLL cells. Collectively, HES1 can promote activation of the Notch1 signaling pathway to cause PTEN transcription inhibition and the subsequent expression reduction, thereby promoting the proliferation and inhibiting the apoptosis of CLL cells.

ACY-1215 suppresses the proliferation and induces apoptosis of chronic myeloid leukemia cells via the ROS/PTEN/Akt pathway.

Chronic myeloid leukemia (CML) is a hematological tumor marked by the bcr-abl fusion gene formed by t (9;22) (q34; q11), which translated into the BCR-ABL protein. Tyrosine kinase inhibitors (TKIs) have been widely used to cure CML patients. Nevertheless, the emergence of TKI resistance has become the problem to the outcome of CML patients. Histone deacetylase 6 (HDAC6), a kind of Hsp90α deacetylase, was detected to be overexpressed in chronic myeloid leukemia stem cells. Besides, the loss of HDAC6 enzymatic activity can result in the degradation of Hsp90α's client proteins, such as BCR-ABL, the oncoprotein of CML. Here, we explored the expression of HDAC6 and discovered that it was upregulated compared with control in CML. Then we explored the effect of Rocilinostat (ACY-1215), a specific HDAC6 inhibitor, on CML cells. Our results proved that ACY-1215 could induce apoptosis and cell cycle arrest in a ROS-dependent manner. Moreover, we detected a downregulation of the BCR-ABL signaling pathway in the ACY-1215 treatment group. Mechanistically, we noted that the upregulation of PTEN was induced after being treated by ACY-1215 and its downstream protein p-Akt was decreased. The Akt activator SC79 can partially reverse the influence of ACY-1215 on CML cells. Besides, our results also proved that ACY-1215 can synergize with imatinib to suppress chronic myeloid leukemia in vitro and in vivo. On the whole, our study revealed that HDAC6 is a possible therapeutic target in CML, and the combination therapy of TKI and HDAC6 inhibitor may improve the outcome of CML patients.

One Stone, Two Birds: N6-Methyladenosine RNA Modification in Leukemia Stem Cells and the Tumor Immune Microenvironment in Acute Myeloid Leukemia.

Acute myeloid leukemia is the most common acute leukemia in adults, with accumulation of abundant blasts and impairment of hematogenic function. Despite great advances in diagnosis and therapy, the overall survival of patients with acute myeloid leukemia remains poor. Leukemia stem cells are the root cause of relapse and chemoresistance in acute myeloid leukemia. The tumor immune microenvironment is another trigger to induce recurrence and drug resistance. Understanding the underlying factors influencing leukemia stem cells and the tumor immune microenvironment is an urgent and unmet need. Intriguingly, N6-methyladenosine, the most widespread internal mRNA modification in eukaryotes, is found to regulate both leukemia stem cells and the tumor immune microenvironment. Methyltransferases and demethylases cooperatively make N6-methyladenosine modification reversible and dynamic. Increasing evidence demonstrates that N6-methyladenosine modification extensively participates in tumorigenesis and progression in various cancers, including acute myeloid leukemia. In this review, we summarize the current progress in studies on the functions of N6-methyladenosine modification in acute myeloid leukemia, especially in leukemia stem cells and the tumor immune microenvironment. We generalize the landscape of N6-methyladenosine modification in self-renewal of leukemia stem cells and immune microenvironment regulation, as well as in the initiation, growth, proliferation, differentiation, and apoptosis of leukemia cells. In addition, we further explore the clinical application of N6-methyladenosine modification in diagnosis, prognostic stratification, and effect evaluation. Considering the roles of N6-methyladenosine modification in leukemia stem cells and the tumor immune microenvironment, we propose targeting N6-methyladenosine regulators as one stone to kill two birds for acute myeloid leukemia treatment.

Paeoniflorin induces apoptosis and cycle arrest in B-cell acute lymphoblastic leukemia cells by inhibiting SENP1/c-Myc signaling pathway

The effect of paeoniflorin on apoptosis and cell cycle in human B-cell acute lymphoblastic leukemia(B-ALL) and its underlying mechanism were investigated in this study. Nalm-6 and SUP-B15 cells were cultured in vitro and divided into control group(0 μg·mL~(-1)) and experimental groups(200, 400, and 800 μg·mL~(-1) paeoniflorin). Cell counting kit-8(CCK-8) was used to measure the viability of Nalm-6 and SUP-B15 cells, and cell apoptosis and cell cycle distribution were analyzed by flow cytometry. Western blot was used to detect the protein levels of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase(cleaved PARP), c-Myc, and small ubiquitin-like modifier-specific protease 1(SENP1). The mRNA levels of c-Myc and SENP1 in acute lymphoblastic leukemia(ALL) patients were analyzed based on the Oncomine database. AutoDock was used for molecular docking to analyze the interaction of paeoniflorin with c-Myc and SENP1 proteins. RESULTS:: showed that paeoniflorin inhibited the viability of Nalm-6 and SUP-B15 cells in concentration and time-dependent manners. Compared with the control group, paeoniflorin significantly up-regulated the expression of apoptosis-related proteins cleaved caspase-3 and cleaved PARP to induce apoptosis, evidently increased the proportion of G_2/M phase cells and induced G_2/M phase arrest, and obviously down-regulated the expression of c-Myc and SENP1 proteins in Nalm-6 and SUP-B15 cells. The mRNA levels of c-Myc and SENP1 in ALL patients were higher than those in the normal cell. Molecular docking demonstrated that paeoniflorin had good binding to c-Myc and SENP1 proteins. In summary, paeoniflorin inhibits the proliferation of Nalm-6 and SUP-B15 cells by inducing apoptosis and G_2/M phase arrest, which may be related to the down-regulation of c-Myc and SENP1 proteins.

Twist1 Promoter Methylation Regulates the Proliferation and Apoptosis of Acute Myeloid Leukemia Cells via PI3K/AKT Pathway.

Twist-related protein 1 (Twist1) is a widely recognized oncogene in acute myeloid leukemia (AML), and its promoter methylation is related with the progression of solid tumors. However, the association between Twist1 promoter methylation and AML has not been well studied. Twist1 mRNA expression was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of Twist1 and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signal were measured via western blotting. Methylation-specific PCR was performed to detect the methylation status of Twist1 promoter. CCK-8 assay and flow cytometry were used to reveal cellular biological effects. Twist1 expression and promoter methylation level were significantly upregulated in AML tissues and cell lines and were further downregulated in demethylating agent 5'-azacitidine (5-Aza)-treated cells. Ectopic expression of Twist1 increased AML cell viability, while reducing apoptosis, and attenuated the effects of 5-Aza on the proliferation and apoptosis. We also found that the PI3K/AKT signaling pathway was positively regulated by Twist1. Our findings revealed that Twist1 accelerates the tumorigenesis of AML cells by promoting its promoter methylation via the activation of PI3K/AKT signaling pathway.

Effect of Curcumin on Apoptosis of Acute T-Lymphoblastic Leukemia Cells induced by UMI-77 and Its Related Mechanism

T-ALL cell line Molt-4 was cultured, and the cells were treated with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77 for 24 h. The MTT method was used to detect the cell survival rate after different treatment; According to the results of curcumin and UMI-77, the experimental settings were divided into control group, curcumin group (20 μmol/L curcumin treated cells), UMI-77 group (15 μmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells) and curcumin+ UMI-77 group (20 μmol/L curcumin and 15 μmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells), MTT method was used to detect cell proliferation inhibition rate, Annexin V-FITC/PI double staining method and TUNEL staining were used to detect cell apoptosis, DCFH-DA probe was used to detect cell reactive oxygen species, JC-1 fluorescent probe was used to detect mitochondrial membrane potential, Western blot was used to detect the expression levels of apoptosis-related proteins and Notch1 signaling pathway-related proteins. After the treatment of Molt-4 cells with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77, the cell survival rate was decreased (P<0.05); Compared with the control group, the cell proliferation inhibition rate of the curcumin group and the UMI-77 group were increased, the apoptosis rate of cell was increased, the level of ROS was increased, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, and the protein expression of Bcl-2 was reduced (P<0.05); Compared with the curcumin group or UMI-77 group, the cell proliferation inhibition rate and apoptosis rate of the curcumin+UMI-77 group were further increased, and the level of ROS was increased. At the same time, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, the protein expression of Bcl-2 was reduced (P<0.05); In addition, the mitochondrial membrane potential of the cells after curcumin treatment was decreased, and the proteins expression of Notch1 and HES1 were reduced (P<0.05). Curcumin can enhance the apoptosis of T-ALL cells induced by Mcl-1 small molecule inhibitor UMI-77 by reducing the mitochondrial membrane potential, the mechanism may be related to the inhibition of Notch1 signaling pathway.