Acute Myeloid Leukemia Cells Research Articles

Discovery of novel and highly potent small molecule inhibitors targeting FLT3-ITD for the treatment of acute myeloid leukemia using structure-based virtual screening and biological evaluation

Considering the essential role of FLT3-ITD mutations in the development of acute myeloid leukemia (AML), the research and development of FLT3 inhibitors hold significant therapeutic potential. In this study, we identified a novel, highly potent small molecule inhibitor, FLIN-4, targeting FLT3 through structure-based virtual screening. Notably, FLIN-4 showed exceptional inhibitory effects in kinase activity inhibition assays, exhibiting a potent inhibitory effect against FLT3 (IC50 = 1.07 ± 0.04 nM). This potency was significantly superior to that of the known positive inhibitor Midostaurin, showing approximately 27 times higher inhibitory potency. Molecular dynamics simulations have confirmed the stable interaction between FLIN-4 and FLT3. Furthermore, cytotoxicity assays revealed that FLIN-4 has significant anti-proliferative activity against the AML cell line MV4-11 (IC50 = 1.31 ± 0.06 nM). Overall, these data suggest that FLIN-4, as a potential therapeutic candidate for AML, is valuable for further research and development.

Discovery of novel XPO1 PROTAC degraders for the treatment of acute myeloid leukemia.

Targeting XPO1 inhibition has emerged as a promising therapeutic strategy in cancer treatment. Despite the numerous XPO1 inhibitors reported to date, no XPO1 degraders have been disclosed. In this study, we reported the design, synthesis and biological characterization of small-molecule XPO1 degraders based upon the proteolysis targeting chimera (PROTAC), marking the first public disclosure of XPO1 degraders. The potent PROTAC compound 2c was identified, demonstrating effective degradation of XPO1 protein in MV4-11 acute myeloid leukemia (AML) cells, with a DC50 of 23.67nM. Treatment with 2c resulted in significant antiproliferative effects, with IC50 values of 0.142±0.029μM in MV4-11cells and 0.186±0.024μM in MOLM-13cells. Additionally, 2c induced apoptosis, inhibited NF-κB activity, and caused G1 phase cell cycle arrest. The study highlights the therapeutic potential of targeting XPO1 degradation in AML treatment and emphasizes the advantages of PROTAC technology in developing novel anticancer strategies. These findings provide a foundation for further exploration of XPO1 degraders in cancer therapy, offering new hope for effective treatment options in hematological malignancies.

Establishment of a high-risk pediatric AML-derived cell line YCU-AML2 with genetic and metabolic vulnerabilities.

The prognosis of acute myeloid leukemia (AML) with KMT2A::MLLT3 rearrangement and MECOM overexpression and/or KRAS mutation is dismal, and the optimal treatment strategy remains unclear. However, to the best of our knowledge, a suitable model (such as a cell line or its xenograft model) for research on this subtype has not been established. We established a novel AML cell line, YCU-AML2, and its xenograft model harboring KMT2A::MLLT3 rearrangement, MECOM overexpression, and KRAS G12A mutation. YCU-AML2 xenograft mice models developed AML and mimicked the clinical phenotype of the original patient. YCU-AML2 expressed high sensitivity to MEK inhibitors, such as trametinib and selumetinib. Moreover, YCU-AML2 also exhibited high sensitivity to L-asparaginase with glutaminase activity, perhaps because of its reliance on oxidative phosphorylation via glutaminolysis as its main energy source. We believe that the YCU-AML2 cell line and its xenograft model can serve as models to explore the molecular pathogenesis of high-risk AML with KMT2A::MLLT3 rearrangement, MECOM overexpression, and/or KRAS mutation and develop new treatment strategies.

Anlotinib enhances the pro-apoptotic effect of APG-115 on acute myeloid leukemia cell lines by inhibiting the P13K/AKT signaling pathway.

APG-115 is a novel small-molecule selective inhibitor that destabilizes the p53-MDM2 complex and activates p53-mediated apoptosis in tumor cells. Anlotinib inhibits tumor angiogenesis and promotes apoptosis. In this study, we investigated the apoptotic effect and potential mechanism of APG-115 and anlotinib combination on AML cell lines with different p53 backgrounds. The IC50 values ​of APG-115 and anlotinib were detected by CCK-8 assay. The apoptosis rate of AML cells was evaluated by Annexin-V and PI double staining. Transcriptome sequencing was performed on the MOLM16 cell line treated with APG-115 and anlotinib, and differential analysis and enrichment analysis were performed. Real-time quantitative PCR and Western blot were used to detect the changes in cell cycle and pathway-related genes and proteins in AML cell lines after drug treatment. In vivo experiments, the anti-leukemia effects of APG-115 and anlotinib on AML xenograft mouse models were evaluated. APG-115 and anlotinib could independently promote AML cell apoptosis, and the combination of the two drugs could produce a synergistic effect. Transcriptome sequencing showed that compared with the APG-115 monotherapy group, the differentially expressed genes were mainly enriched in the MDM2-p53 and PI3K/AKT pathways. In vivo experiments showed that compared with AML xenograft mice treated with either drug alone, AML progression was slowed in AML xenograft mice treated with APG-115 and anlotinib. In vivo and in vitro experimental have shown that APG-115 combined with anlotinib can promote AML cells apoptosis and inhibit the progression of disease is independent of the p53 status.

Novel antileukemic compound with sub-micromolar potency against STAT5 addicted myeloid leukemia cells.

Signal Transdcer and Activator of Transcription 5A and 5B (STAT5A/5B) are key effectors of tyrosine kinase oncogenes in myeloid leukemias. It is now clearly evidenced that inhibition of STAT5A/5B not only blocks the growth and survival of myeloid leukemia cells but also overcomes the resistance of leukemic cells to chemotherapy. Previous screening experiments allowed us to identify 17f as a lead compound with promising antileukemic activity that blocks the phosphorylation and transcriptional activity of STAT5A/5B in myeloid leukemia cells addicted to these proteins. In light of these findings, we initiated further pharmacomodulations of 17f to develop new derivatives with enhanced antileukemic activity. Our screening assays identified 14a, an aminopyrimidine derivative of 17f, as a new lead compound that: 1) blocks the growth and survival of myeloid leukemia cells at sub-micromolar concentrations, 2) targets the phosphorylation of STAT5 but also the expression of STAT5B and 3) relieves the resistance of Chronic and Acute Myeloid leukemia cells to conventional chemotherapy.

Discovery of 5-imidazole-3-methylbenz[d]isoxazole derivatives as potent and selective CBP/p300 bromodomain inhibitors for the treatment of acute myeloid leukemia.

Inhibition of the bromodomain of the cAMP response element binding protein (CREB)-binding protein (CBP) and its homologue p300 is an attractive therapeutic approach in oncology, particularly in acute myeloid leukemia (AML). In this study we describe the design, optimization, and evaluation of 5-imidazole-3-methylbenz[d]isoxazoles as novel, potent and selective CBP/p300 bromodomain inhibitors. Two of the representative compounds, 16t (Y16524) and 16u (Y16526), bound to the p300 bromodomain with IC50 values of 0.01 and 0.03 μM, respectively. Furthermore, 16t and 16u potently inhibited the growth of AML cell lines, particularly MV4;11 cells with IC50 values of 0.49 and 0.26 μM, respectively. The potent CBP/p300 bromodomain inhibitors represent a new class of compounds for the development of potential therapeutics against AML.

Concurrent inhibition of p300/CBP and FLT3 enhances cytotoxicity and overcomes resistance in acute myeloid leukemia.

FMS-like tyrosine kinase-3 (FLT3), a class 3 receptor tyrosine kinase, can be activated by mutations of internal tandem duplication (FLT3-ITD) or point mutations in the tyrosine kinase domain (FLT3-TKD), leading to constitutive activation of downstream signaling cascades, including the JAK/STAT5, PI3K/AKT/mTOR and RAS/MAPK pathways, which promote the progression of leukemic cells. Despite the initial promise of FLT3 inhibitors, the discouraging outcomes in the treatment of FLT3-ITD-positive acute myeloid leukemia (AML) promote the pursuit of more potent and enduring therapeutic approaches. The histone acetyltransferase complex comprising the E1A binding protein P300 and its paralog CREB-binding protein (p300/CBP) is a promising therapeutic target, but the development of effective p300/CBP inhibitors faces challenges due to inherent resistance and low efficacy, often exacerbated by the absence of reliable clinical biomarkers for patient stratification. In this study we investigated the role of p300/CBP in FLT3-ITD AML and evaluated the therapeutic potential of targeting p300/CBP alone or in combination with FLT3 inhibitors. We showed that high expression of p300 was significantly associated with poor prognosis in AML patients and positively correlated with FLT3 expression. We unveiled that the p300/CBP inhibitors A485 or CCS1477 dose-dependently downregulated FLT3 transcription via abrogation of histone acetylation in FLT3-ITD AML cells; in contrast, the FLT3 inhibitor quizartinib reduced the level of H3K27Ac. Concurrent inhibition of p300/CBP and FLT3 enhanced the suppression of FLT3 signaling and H3K27 acetylation, concomitantly reducing the phosphorylation of STAT5, AKT, ERK and the expression of c-Myc, thereby leading to synergistic antileukemic effects both in vitro and in vivo. Moreover, we found that p300/CBP-associated transcripts were highly expressed in quizartinib-resistant AML cells with FLT3-TKD mutation. Targeting p300/CBP with A485 or CCS1477 retained the efficacy of quizartinib, suggesting marked synergy when combined with p300/CBP inhibitors in quizartinib-resistant AML models, as well as primary FLT3-ITD+ AML samples. These results demonstrate a potential therapeutic strategy of combining p300/CBP and FLT3 inhibitors to treat FLT3-ITD and FLT3-TKD AML.

P-1759. Antibiotic De-Escalation in Neutropenic Fever

Abstract Background Cancer patients with neutropenic fever are frequently continued on broad-spectrum empiric antimicrobial therapy (EAT) until neutrophil recovery, but recent randomized trials demonstrate that EAT de-escalation prior to neutrophil recovery is safe in certain subgroups. We evaluated outcomes of EAT de-escalation based on clinical criteria in patients with neutropenic fever at our institution. Methods We retrospectively reviewed charts of 49 patients with acute myeloid leukemia (AML) or hematologic stem cell transplantation (HSCT) and neutropenic fever following revisions to Standard of Practice (SOP) institutional guidelines (hereafter “SOP” group) allowing for antibiotic de-escalation using a clinical approach in comparison to a historic cohort of 60 patients. The revised SOP recommended antibiotic de-escalation to levofloxacin prophylaxis in patients who received ≥ 72 hours of EAT, were afebrile and clinically stable for 48 hours, and did not have a clinically or microbiologically documented infection. The primary endpoint was the EAT duration. Secondary end points included incidence of recurrent fever, infections, vasopressor support and/or intensive care unit (ICU) stay, and in-hospital mortality. Results Baseline characteristics were similar between both groups, with differences including a larger proportion of males in the SOP group compared to the control group (59% vs. 35%) and the primary underlying malignancy of multiple myeloma in the SOP group compared to AML in the control group. The median duration of EAT prior to de-escalation was shorter in the SOP group compared to the historic cohort (5.0 [IQR 4.0, 6.0] vs. 7.0 days [IQR 5.0, 10.2], p< 0001). There was no difference in incidence of recurrent fever (20% vs. 30%, p=0.3) or secondary infection (10% vs. 12%, p=0.8). Incidence of vasopressor support and/or ICU stay(10% vs. 3.3%, p=0.2) and in-hospital mortality (2.0% vs. 3.3 %, p >0.9) were also similar between both groups. Conclusion Our study indicates that our new institutional guidelines successfully lead to reduced broad spectrum antimicrobial exposure without increased infections, ICU stays, or mortality. Thus, de-escalation of EAT based on clinical criteria is safe in this high risk patient population. Disclosures Douglas Tremblay, MD, AbbVie: Advisor/Consultant|Astellas: Grant/Research Support|Cogent Biosciences: Advisor/Consultant|Cogent Biosciences: Grant/Research Support|Gilead: Grant/Research Support|GSK: Advisor/Consultant|Novartis: Advisor/Consultant|Sierra Oncology: Advisor/Consultant|Sobi: Advisor/Consultant|Sobi: Grant/Research Support|Sobi: Patent for use of pacritinib and azacitidine in CMML|Sumitomo: Grant/Research Support Samantha E. Jacobs, MD, MS, Ansun Biopharma: Advisor/Consultant|Eurofins, Viracor, LLC.: Grant/Research Support

Allogeneic DNT cell therapy synergizes with T cells to promote anti-leukemic activities while suppressing GvHD

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a second-line treatment with curative potential for leukemia patients. However, the prognosis of allo-HSCT patients with disease relapse or graft-versus-host disease (GvHD) is poor. CD4+ or CD8+ conventional T (Tconv) cells are critically involved in mediating anti-leukemic immune responses to prevent relapse and detrimental GvHD. Hence, treatment for one increases the risk of the other. Thus, therapeutic strategies that can address relapse and GvHD are considered the Holy Grail of allo-HSCT. CD3+CD4−CD8− double-negative T cells (DNTs) are unconventional mature T cells with potent anti-leukemia effects with “off-the-shelf” potential. A phase I clinical trial demonstrated the feasibility, safety, and potential efficacy of allogeneic DNT therapy for patients with relapsing acute myeloid leukemia (AML) post-allo-HSCT. Here, we studied the impact of DNTs on the anti-leukemic and GvHD-inducing activities of Tconv cells. DNTs synergized with Tconv cells to mediate superior anti-leukemic activity. Mechanistically, DNTs released soluble factors which activated and evoked potent anti-leukemic activities of Tconv cells. In contrast, DNTs suppressed GvHD-inducing activities of Tconv cells in a CD18-dependent manner by mediating cytotoxicity against proliferative Tconv cells. The seemingly opposite immunological activities of DNTs were dictated by the presence or absence of AML cells. Collectively, these results support the potential of DNTs as an adjuvant to allo-HSCT to address both disease relapse and GvHD.

Screening potential antileukemia ingredients from sweet potato: integration of metabolomics analysis, network pharmacology, and experimental validation

BackgroundActive dietary flavonoids are a promising resource for novel drug discovery. Sweet potato, a widely cultivated functional crop, is abundant in flavonoids. However, the active ingredients associated with acute myeloid leukemia (AML) treatment and their underlying mechanisms have not been reported to date.ObjectiveThis study aims to identify novel drugs against AML from sweet potato by integrating metabolomics analysis, network pharmacology, and experimental validation.MethodsFirstly, ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was employed to analyze the major constituents in sweet potato. Then, nine active ingredients were selected for validation of their anti-leukemia effects. Subsequently, three of them underwent network pharmacology analyses and in vitro experimental verification. Finally, the anti-leukemia effect of cynaroside was further confirmed through in vivo experimental validation.ResultsFirstly, the flavonoid content of stem, leaves, flesh, and peel from 13 sweet potato cultivars was examined. The leaves of Nanshu 017 exhibited the highest flavonoid content of 2.27% dry weight (DW). Then, an extract derived from these leaves was employed for in vitro experiments, demonstrating significant inhibition of AML cell growth. Subsequently, based on the results of metabolomics analysis and network pharmacology, cynaroside, nepitrin, and yuanhuanin were identified as potential antileukemia agents present in sweet potato for the first time; while CASP3, KDR, EGFR, and SRC were recognized as pivotal targets of these three monomers against AML. Finally, the antileukemia effects of cynaroside, nepitrin, and yuanhuanin were confirmed through in vitro and in vivo experimental validation.ConclusionIn summary, sweet potato leaves extract possesses an antileukemic effect while cynaroside, nepitrin, and yuanhuanin demonstrate potential as treatments for AML.

Integrated Genomics Reveal Potential Resistance Mechanisms of PANoptosis-Associated Genes in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is marked by the proliferation of abnormal myeloid progenitor cells in the bone marrow and blood, leading to low cure rates despite new drug approvals from 2017 to 2018. Current therapies often fail due to the emergence of drug resistance mechanisms, such as those involving anti-apoptotic pathways and immune evasion, highlighting an urgent need for novel approaches to overcome these limitations. Programmed cell death (PCD) is crucial for tissue homeostasis, with PANoptosis-a form of PCD integrating pyroptosis, apoptosis, and necroptosis-recently identified. This process, regulated by the PANoptosome complex, could be key to overcoming AML drug resistance. Targeting multiple PCD pathways simultaneously may prove more effective than single-target therapies. Research suggests that disrupting anti-apoptotic mechanisms, such as those involving Bcl-2, can enhance drug sensitivity in AML. This study hypothesizes that PANoptosis-associated resistance genes (PARGs) play a critical role in AML drug resistance by modulating immune responses and offers a multi-faceted approach to tackle this challenge. Using RNA sequencing data from the Cancer Genome Atlas and Gene Expression Omnibus databases, we performed differential expression analysis to identify significantly dysregulated PARGs in AML. Regression analysis identified prognostic PARGs, bridging a key gap in understanding how these genes contribute to treatment resistance. We then verified their expression in AML cell lines and cell samples treated with cytarabine using RT-qPCR. Hierarchical clustering revealed distinct PARG expression patterns, and functional enrichment analysis highlighted their involvement in immune-related pathways. The combination of bioinformatics and experimental validation underscores how these genes may mediate immune modulation in drug resistance, providing a robust framework for further study. Our findings suggest that PARGs contribute to AML resistance by modulating immune responses and provide potential targets for therapeutic intervention. This study highlights the potential of targeting PARGs to improve treatment outcomes in AML. By analyzing the expression changes of these genes in response to standard clinical treatments, we provide a framework for developing multi-target therapeutic strategies that simultaneously disrupt multiple programmed cell death pathways. Such an approach directly addresses the limitations of current treatments by offering a method to enhance drug sensitivity and mitigate resistance, potentially improving survival rates. Our findings underscore the importance of a comprehensive understanding of PCD mechanisms and pave the way for innovative treatments that could significantly impact AML management.

Effective eradication of acute myeloid leukemia stem cells with FLT3-directed antibody-drug conjugates.

Refractory disease and relapse are major challenges in acute myeloid leukemia (AML) therapy attributed to survival of leukemic stem cells (LSC). To target LSCs, antibody-drug conjugates (ADCs) provide an elegant solution, combining the specificity of antibodies with highly potent payloads. We aimed to investigate if FLT3-20D9h3-ADCs delivering either the DNA-alkylator duocarmycin (DUBA) or the microtubule-toxin monomethyl auristatin F (MMAF) can eradicate quiescent LSCs. We show here that DUBA more potently kills cell-cycle arrested AML cells compared to microtubule-targeting auristatins. Due to limited stability of 20D9h3-DUBA ADC in vivo, we analyzed both ADCs in advanced in vitro stem cell assays. 20D9h3-DUBA successfully eliminated leukemic progenitors in vitro in colony-forming unit and long-term culture initiating cell assays, both in patient cells and in patient-derived xenograft (PDX) cells. Further, it completely prevented engraftment of AML PDX leukemia-initiating cells in NSG mice. 20D9h3-MMAF had a similar effect in engraftment assays, but a less prominent effect in colony assays. Both ADCs did not affect healthy stem and progenitor cells at comparable doses providing the rationale for FLT3 as therapeutic LSC target. Collectively, we show that FLT3-directed ADCs with DUBA or MMAF have potent activity against AML LSCs and represent promising candidates for further clinical development.

XPO1/Exportin-1 in Acute Myelogenous Leukemia; Biology and Therapeutic Targeting

Exportin 1 is responsible for the export of hundreds of proteins, several RNA species and ribosomal components from the nucleus to the cytoplasm. Several transported proteins are important for regulation of cell proliferation and survival both in normal and malignant cells. We review the biological importance and the possibility of therapeutic targeting of Exportin 1 in acute myeloid leukemia (AML). Exportin 1 levels can be increased in human primary AML cells, and even exportin inhibition as monotherapy seems to have an antileukemic effect. The results from Phase I/II studies also suggest that exportin inhibition can be combined with conventional chemotherapy, including intensive induction and consolidation therapy possibly followed by allogeneic stem cell transplantation as well as AML-stabilizing therapy in elderly/unfit patients with hypomethylating agents. However, the risk of severe toxicity needs to be further evaluated; hematological toxicity is common together with constitutional side effects, electrolyte disturbances, and gastrointestinal toxicity. A recent randomized study of intensive chemotherapy with and without the Exportin inhibitor selinexor in elderly patients showed reduced survival in the selinexor arm; this was due to a high frequency of relapse and severe infections during neutropenia. Experimental studies suggest that Exportin 1 inhibition can be combined with other forms of targeted therapy. Thus, Exportin 1 inhibition should still be regarded as a promising strategy for AML treatment, but future studies should focus on the risk of toxicity when combined with conventional chemotherapy, especially in elderly/unfit patients, combinations with targeted therapies, identification of patient subsets (AML is a heterogeneous disease) with high susceptibility, and the possible use of less toxic next-generation Exportin 1 inhibitors.

Bispecific Aptamer-Drug Conjugates Selectively Eliminate Malignant Hematologic Cells for Treating Acute Myeloid Leukemia.

Surface antigen-directed immunotherapy is a curative treatment modality for acute myeloid leukemia (AML) that is characterized by the abundance and stability expression of surface antigens. However, current surface antigen-directed immunotherapies have shown poor outcomes and undesirable mortality rates in treating AML patients, primarily due to acquired resistance that arises from using single-target therapies to address the heterogeneous expression of surface antigens. Hence, in order to improve the efficacy of antigen-specific therapies for treating AML, we designed a bispecific aptamer-drug conjugate. In particular, cell-SELEX incorporating cell lysate-SELEX for aptamers with HEL cells yielded AptCD117, which specifically binds to CD117 (a highly expressed marker on both hematopoietic stem cells and primary AML cells) and has excellent performance in targeting human AML cells. Combined with CD71-binding aptamer LXD-11b (another broadly expressed surface antigen on leukemia cells), bispecific aptamers were designed to couple with monomethyl auristatin F (MMAF) for fabricating aptamer-drug conjugates. Results demonstrated that bispecific aptamer-MMAF conjugates efficiently kill different CD117 and CD71 expression levels of target AML cell lines in vitro. Importantly, the exposure of AML marrow specimens to bispecific aptamer-MMAF conjugates resulted in the selective elimination of primary AML cells in vitro and had no effect on healthy lymphocytes within the same specimens. Thus, these results provide a proof of concept for the generation of bispecific aptamer-drug conjugates directed against human AML cells, which hold the promise of advancing treatment strategies and improving AML patient outcomes.

Fenretinide targets GATA1 to induce cytotoxicity in GATA1 positive Acute Erythroid and Acute Megakaryoblastic Leukemic cells.

- Fenretinide (4-HPR) targets the transcription factor GATA1, which was previously thought to be "undruggable" and induces GATA1 loss.- M6 and M7 Acute Myeloid Leukemias (AML) have enriched expression of GATA1 and they can be considered GATA1 positive.- Loss of GATA1 contributes significantly to 4-HPR cytotoxicity in M6 OCIM1 cells.- 4-HPR treatment overcomes chemotherapeutic resistance in M6 Acute Myeloid Leukemia cells, synergizes with standard-of-care and outperforms standard-of-care as a single agent.

FHL1 as a prognostic biomarker and therapeutic target in acute promyelocytic leukaemia

Acute myeloid leukemia (AML) has a poor prognosis and high heterogeneity. Most cases of leukemias are caused by environmental factors interacting with the cell’s genetic material, but treatment is still dominated by cell cycle drugs. Therefore, there is an urgent need to find reliable biomarkers. Based on the Gene Expression Omnibus database, Kaplan–Meier survival analysis and univariate Cox regression analysis were used to select the genes that had the most significant influence on the prognosis of patients with AML. Quantitative real-time PCR and Western blot were used to assess the effects of small interfering RNA transfection and lentiviral interference on the gene's knockout and overexpression, respectively. These method were also used to confirm the expression levels of the FHL1 gene in the HL60 cell line compared to neutrophils.. Cell Counting Kit-8 and flow cytometry were used to detect the effect of high or low expression of FHL1 on cell viability and apoptosis under the influence of cytarabine and daunorubicin. FHL1 was found to be the most prognostic independent biomarker by GSE12417 screening and GSE37642 validation. FHL1 is highly expressed in AML, and knockdown of FHL1 can increase the sensitivity of AML cells to cytarabine and daunorubicin. FHL1 may play a role as a potential molecular marker and therapeutic target for predicting poor prognosis of AML and for direct treatment (chemotherapy).

Unveiling cellular changes in leukaemia cell lines after cannabidiol treatment through lipidomics

The present study was aimed at revealing the metabolic changes that occurred in the cellular lipid pattern of acute and chronic myeloid leukaemia cells following treatment with cannabidiol (CBD). CBD is a non-psychoactive compound present in Cannabis sativa L., which has shown an antiproliferative action in these type of cancer cells. CBD treatment reduced cell viability and initiated apoptotic and necrotic processes in both cancer cell lines in a time and dose-dependent manner, showing acute myeloid leukaemia (HL-60) cells greater sensitivity than chronic myeloid leukaemia ones (K-562), without differences in the activation of caspases 3/7. Then, control and treated cells of HL-60 and K-562 cell lines were studied through an untargeted lipidomic approach. The treatment was carried out with CBD at a concentration of 10 μM for HL-60 cells and 23 µM CBD for K-562 cells for 48 h. After the extraction of the lipid content from cell lysates, the samples were analysed by UHPLC-QTOF-MS/MS both in the positive and the negative ionization modes. The comprehensive characterization of cellular lipids unveiled several classes significantly affected by CBD treatment. Most of the differences correspond to phospholipids, including cardiolipins (CL), phosphatidylcholines (PC) and phosphosphingolipids (SM), and also triacylglycerols (TG), being many TG species increased after CBD treatment in the acute and chronic models, whereas phospholipids were found to be decreased. The results highlight some important lipid alterations related to CBD treatment, plausibly connected with different metabolic mechanisms involved in the process of cell death by apoptosis in cancer cell lines.

Hypoxia impairs decitabine-induced expression of HLA-DR in acute myeloid leukaemia cell lines

BackgroundHypomethylating agents (HMA), such as azacytidine (AZA) and decitabine (DAC), are epigenetic therapies used to treat some patients with acute myeloid leukaemia (AML) and myelodysplastic syndrome. HMAs act in a replication-dependent manner to remove DNA methylation from the genome. However, AML cells targeted by HMA therapy are often quiescent within the bone marrow, where oxygen levels are low. In this study, we investigate the effects of hypoxia on HMA responses in AML cells.ResultsAML cell lines (MOLM-13, MV-4-11, HL-60) were treated with DAC (100 nM) or AZA (500–2000 nM) in normoxic (21% O2) and hypoxic (1% O2) conditions. Hypoxia significantly reduced AML cell growth across all cell lines, with no additional effects observed upon HMA treatment. Hypoxia had no impact on the extent of DNA hypomethylation induced by DAC treatment, but limited AZA-induced loss of methylation from the genome. Transcriptional responses to HMA treatment were also altered, with HMAs failing to up-regulate antigen presentation pathways in hypoxia. In particular, cell surface expression of the MHC class II receptor, HLA-DR, was increased by DAC treatment in normoxia, but not hypoxia.ConclusionOur results suggest that HMA-induced antigen presentation may be impaired by hypoxia. This study highlights the need to consider microenvironmental factors when designing co-treatment strategies to improve HMA therapeutic efficacy.

Identification and validation of BATF as a prognostic biomarker and regulator of immune cell infiltration in acute myeloid leukemia.

Basic leucine zipper ATF-like transcription factor (BATF) is a nuclear basic leucine zipper protein affiliated with the AP-1/ATF superfamily. Previous research has confirmed that BATF expression plays a significant role in the tumour microenvironment. However, the associations between BATF expression and prognoses in acute myeloid leukaemia (AML) patients and their immunological effects remain unclear. Genomic and clinical AML data were got from the TCGA (TCGA-LAML) and GEO (GSE37642) databases for the subsequent analysis. The expression levels of BATF in AML patients were assessed using GEPIA, and the results were verified by qRT-PCR and Western blotting. In the meantime, the prognostic value of BATF was evaluated using univariate and multivariate analyses, receiver operating characteristic (ROC) curve (AUC) analysis, and Kaplan-Meier (KM) survival analysis. EdU, colony formation, and CCK-8 assays were employed to evaluate the proliferation of cells. Moreover, we detected the association of BATF expression with drug sensitivity through database analysis and in vivo experiments. To further investigate the mechanism of action of BATF in AML, RNA sequencing (RNA-seq) analysis was performed, followed by pathway enrichment analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Meanwhile, we detected the connection between BATF expression and the proportions of immune cells via flow cytometry in C1498 mouse model of AML. Finally, we investigated the association between BATF expression and cell-cell communication within the AML cell population using single-cell sequencing. In this study, we thoroughly investigated the role of BATF in AML. First, we observed a significant elevation in the expression of BATF in patients and cells in AML. Further analysis revealed an association between high BATF expression and poor prognosis in AML. Additionally, BATF expression was found to promote the proliferative capacity of AML cells. Moreover, the results showed that the expression level of BATF dramatically affected the effect of chemotherapy in AML patients. We also discovered that BATF expression could activate multiple immune-related pathways, altering the proportions of CD8+T cells and NK cells, suggesting that BATF may be a regulator of immune cell infiltration. Finally, there were differences in receptor ligand pairs between AML cells with high and low expression of BATF and immune cells. Bioinformatics analysis and experimental verification revealed that BATF expression could alter the proportions of CD8+T cells and NK cells in AML and affect drug sensitivity, making it a potential treatment target for AML.

ALKBH3-mediated m1A demethylation promotes the malignant progression of acute myeloid leukemia by regulating ferroptosis through the upregulation of ATF4 expression

SUMMARY To investigate the role of ALKBH3 in acute myeloid leukemia (AML), we constructed an animal model of xenotransplantation of AML. Our study demonstrated that ALKBH3-mediated m1A demethylation inhibits ferroptosis in KG-1 cells by increasing ATF4 expression, thus promoting the development of AML. These findings suggest that reducing ALKBH3 expression may be a potential strategy to mitigate AML progression. ABSTRACT Background: Acute myeloid leukemia (AML) is characterized by the unrestrained proliferation of myeloid cells. Studies have shown that ALKBH3 is upregulated in most tumors, but the role of ALKBH3 in AML remains unclear. Methods: In this study, we investigated the function of ALKBH3 in AML cells (KG-1) by immunofluorescence, ELISA, flow cytometry, HE staining, and Western blotting. Results: Our results revealed that ALKBH3 is upregulated in AML and that the downregulation of ALKBH3 inhibited KG-1 cell proliferation and promoted cell apoptosis; at the same time, ALKBH3 upregulated ATF4 expression through m1A demethylation, and the knockdown of ATF4 resulted in increased ferrous iron content; TFR1, ACSL4, and PTGS2 expression; and ROS and MDA levels, whereas SOD and GSH levels and the expression levels of ATF4, SLC7A11, GPX4, and FTH1 decreased in KG-1 cells, thereby promoting ferroptosis. Mechanistically, ALKBH3-mediated m1A demethylation suppressed ferroptosis in KG-1 cells by increasing ATF4 expression, thereby promoting the development of AML. Conclusions: Our study indicated that reducing the expression of ALKBH3 might be a potential target for improving AML symptoms.