Human Leukemia Cell Line Research Articles

β-Catenin Regulates Glycolytic and Mitochondrial Function in T-Cell Acute Lymphoblastic Leukemia

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by a poor prognosis. β-catenin is implicated in the progression of T-ALL, yet the precise mechanisms of β-catenin involvement in the pathogenesis of T-ALL, particularly concerning metabolic processes, remain inadequately elucidated. Methods: A β-catenin knockout cell line was generated in the human leukemic cell line Jurkat using the CRISPR-Cas9 technique. Subsequently, assays were performed to evaluate cell proliferation, apoptosis, and metabolic activity. Comparative transcriptomic analysis was conducted between control cells and β-catenin knockout cells. Finally, a mouse xenograft model was employed to assess whether β-catenin knockout attenuates tumor growth and infiltration in vivo. Results: The deletion of β-catenin significantly inhibited proliferation and induced apoptosis. Additionally, the silencing of β-catenin led to the inhibition of glycolysis and a reduction in both mitochondrial mass and membrane potential. These results indicate that β-catenin may play a crucial role in regulating cell proliferation and apoptosis through the modulation of glycolytic activity and mitochondrial function in T-ALL. Conclusions: In summary, our findings uncover a novel mechanism by which β-catenin influences glycolysis and mitochondrial function in the progression of T-ALL, thereby identifying a potential therapeutic target for patients with relapsed T-ALL.

Sensitivity to Tyrosine Kinase Inhibitors in a Human Philadelphia Chromosome-Positive (Ph+) Leukemia Model With the T315I-Inclusive Compound Mutation.

The T315I-inclusive compound mutation, the multiple mutations including the T315I mutation on the sameBCR::ABL1gene, confers resistance to diverse tyrosine kinase inhibitors (TKIs). Development of the F311I/T315I compound mutation has been reported in chronic myeloid leukemia patients who sequentially showed clinical resistance to imatinib and dasatinib. The establishment of a human leukemia model with the T315I-inclusive compound mutation remains an experimental challenge. Here, we introduced the F311I/T315I compound mutation into the intrinsicBCR::ABL1gene of a human TKI-sensitive Philadelphia chromosome-positive leukemia cell line via homologous recombination using the CRISPR/Cas9 systemand obtained three types of sublines: the F311I mutation alone, the T315I mutation alone, and the F311I/T315I compound mutation. The F311I subline was sensitive to dasatinib but moderately resistant to imatinib and nilotinib, while the T315I subline and the F311I/T315I subline were highly resistant to these TKIs. Notably, the T315I subline and the F311I/T315I subline were sensitive to therapeutic concentrations of ponatinib, although more resistant than the F311I subline. Moreover, the T315 subline and the F311I/T315 subline were sensitive to asciminibat therapeutic concentration, as was the F311I subline. This is the first human leukemia model in which the impact of the T315I-inclusive compound mutation on TKI sensitivity was directly confirmed.

Evi1 governs Kdm6b-mediated histone demethylation to regulate the Laptm4b-driven mTOR pathway in hematopoietic progenitor cells.

Ecotropic viral integration site 1 (EVI1/MECOM) is frequently upregulated in myeloid malignancies. Here, we present an Evi1-transgenic mouse model with inducible expression in hematopoietic stem/progenitor cells (HSPCs). Upon induction of Evi1 expression, mice displayed anemia, thrombocytopenia, lymphopenia, and erythroid and megakaryocyte dysplasia with a significant expansion of committed myeloid progenitor cells, resembling human myelodysplastic syndrome/myeloproliferative neoplasm-like (MDS/MPN-like) disease. Evi1 overexpression prompted HSPCs to exit quiescence and accelerated their proliferation, leading to expansion of committed myeloid progenitors while inhibiting lymphopoiesis. Analysis of global gene expression and Evi1 binding site profiling in HSPCs revealed that Evi1 directly upregulated lysine demethylase 6b (Kdm6b). Subsequently, Kdm6b-mediated H3K27me3 demethylation resulted in activation of various genes, including Laptm4b. Interestingly, KDM6B and LAPTM4B are positively correlated with EVI1 expression in patients with MDS. The EVI1/KDM6B/H3K27me3/LAPTM4B signaling pathway was also identified in EVI1hi human leukemia cell lines. We found that hyperactivation of the LAPTM4B-driven mTOR pathway was crucial for the growth of EVI1hi leukemia cells. Knockdown of Laptm4b partially rescued Evi1-induced abnormal hematopoiesis in vivo. Thus, our study establishes a mouse model to investigate EVI1hi myeloid malignancies, demonstrating the significance of the EVI1-mediated KDM6B/H3K27me3/LAPTM4B signaling axis in their maintenance.

Inflammatory Response of THP1 and U937 Cells: The RNAseq Approach.

THP1 and U937 are monocytic cell lines that are common bioassays to reflect monocyte and macrophage activities in inflammation research. However, THP-1 is a human monocytic leukemia cell line, and U937 originates from pleural effusion of histiocytic lymphoma; thus, even though they serve as bioassay in inflammation research, their response to agonists is not identical. Consequently, there has yet to be a consensus about the panel of strongly regulated genes in THP1 and U937 cells representing the inflammatory response to LPS and IFNG. Therefore, we have performed an RNAseq of THP1 and U937 exposed to LPS and IFNG to identify the most sensitive genes and the unique properties of each individual cell line. When applying a highly stringent threshold, we could identify 43, 8 up and 94, 103 down-regulated genes in THP1 and U937 cells, respectively. In THP1 cells, among the most strongly up-regulated genes are CCL1, CXCL2, CXCL3, IL1A, IL1B, IL6, and PTGES. In U937 cells, the strongest up-regulated genes include CSF2, CSF3, CXCL2, CXCL5, CXCL6, IL1A, IL19, IL36G, IL6, ITGA1, ITGA2, and PTGS2. Even though THP1 is considerably less responsive than U937, there are genes commonly upregulated by LPS and IFNG, including the CCL1, CCL3, CCL20, CXCL2, CXCL3, CXCL8, as well as IL1A, IL1B, IL23A, IL6, and genes of prostaglandin synthesis PTGES and PTGS2. Downregulated genes are limited to NRGN and CD36. This head-to-head comparison revealed that THP1 is less responsive than U937 cells to LPS and IFNG and identified a panel of highly regulated genes that can be applied in bioassays in inflammation research. Our data further propose bulk RNAseq as a standard method in bioassay research.

Integrating eugenol with intensive care in leukemia patients: exploration of pro-apoptotic potential against HL-60, human leukemia cell line

Objective: The objective of this in-vitro study was to explore the pro-apoptotic potential of eugenol (4-allyl-2-methoxyphenol) on Human Leukemia-60 (HL-60) cell line as a potent phytochemical in intensive care setting to leukemia patients. Methodology: After formal approval by all of the respective ethical committees, this study was simultaneously conducted at all respective institutional departments. The study included culturing HL-60 cell line and its treatment with serial concentrations of eugenol for calculation of subsequent IC50 values (half-maximal inhibitory concentration) via MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, expression analysis of gene, comparative analysis of relative gene fold (Intrinsic Biomarkers Caspase-3, Caspase-9) as apoptosis mediator markers followed by RT-qPCR. Cellular apoptotic morphology was confirmed via Hoechst 333258 staining. Results: For HL-60 cell line, the IC50 of eugenol (14.1 uM) showed high gene expression of pro-apoptotic biomarkers (Caspase-3 and Caspase-9). Hoechst 333258 staining showed prominent apoptotic bodies leading to nuclear fragmentations. Conclusion: Eugenol proved to possess robust pro-apoptotic potential leading to diagnostic efficacy against leukemia HL-60 cell line. Further studies would help in identifying key mechanisms by which eugenol exhibits anti-cancer potential against HL-60 cell lines. Abbreviations: HL-60 - Human Leukemia-60; IC50 - Half-Maximal Inhibitory Concentration Keywords: Eugenol, Pro-apoptotic Effects, Intensive Care, Anti-Cancer Mechanisms, HL-60, Natural Chemotherapeutics Citation: Khaliq HMH, Bughio R, Nangdev P, Aziz O, Javed W. Integrating eugenol with intensive care in leukemia patients: exploration of pro-apoptotic potential against HL-60, human leukemia cell line. Anaesth. pain intensive care 2024;28(5):871−875; DOI: 10.35975/apic.v28i5.2491 Received: June 18, 2024; Reviewed: August 03, 2024; Accepted: August 11, 2024

LINC01270 Regulates the NF-κB-Mediated Pro-Inflammatory Response via the miR-326/LDOC1 Axis in THP-1 Cells.

Long intergenic noncoding (LINC)01270 is a 2278 bp transcript belonging to the intergenic subset of long noncoding (lnc)RNAs. Despite increased reports of LINC01270's involvement in different diseases, evident research on its effects on inflammation is yet to be achieved. In the present study, we investigated the potential role of LINC01270 in modulating the inflammatory response in the human monocytic leukemia cell line THP-1. Lipopolysaccharide treatment upregulated LINC01270 expression, and siRNA-mediated suppression of LINC01270 enhanced NF-κB activity and the subsequent production of cytokines IL-6, IL-8, and MCP-1. Interestingly, the knockdown of LINC01270 downregulated expression of leucine zipper downregulated in cancer 1 (LDOC1), a novel NF-κB suppressor. An analysis of the LINC01270/micro-RNA (miRNA)/protein interactome profile identified miR-326 as a possible mediator. Synthetic RNA agents that perturb the interaction among LINC01270, miR-326, and LDOC1 mRNA mitigated the changes caused by LINC01270 knockdown in THP-1 cells. Additionally, a luciferase reporter assay in HEK293 cells further confirmed that LINC01270 knockdown enhances NF-κB activation, while its overexpression has the opposite effect. This study provides insight into LINC01270's role in modulating inflammatory responses to lipopolysaccharide stimulation in THP-1 cells via the miR-326/LDOC1 axis, which negatively regulates NF-κB activation.

Targeting Non-Apoptotic Pathways with the Cell Permeable TAT-Conjugated NOTCH1 RAM Fragment for Leukemia and Lymphoma Cells.

Targeting nonapoptotic cell death offers a promising strategy for overcoming apoptosis resistance in cancer. In this study, we developed Tat-Ram13, a 25-mer peptide that fuses the NOTCH1 intracellular domain fragment RAM13 with a cell-penetrating HIV-1 TAT, for the treatment of T-cell acute lymphoblastic leukemia with aberrant NOTCH1 mutation. Tat-Ram13 significantly downregulated NOTCH1-target genes in T-ALL cell lines. Furthermore, the peptide had potent cytotoxic effects on various human leukemia and lymphoma cell lines. However, it did not affect normal lymphocytes and monocytes, some subsets of leukemia cells, or adherent tumor cells. This cell-selective cytotoxic activity was closely correlated with the peptide uptake via macropinocytosis in leukemia cells. In leukemia cells, Tat-Ram13 triggered rapid cell death. This cell death involved mitochondrial membrane depolarization and extracellular release of lactate dehydrogenase and high-mobility group box-1 protein without activation of caspase-3 or cleavage of PARP-1. These results suggest that Tat-Ram13 cell death is nonapoptotic and mediated by rapid plasma membrane rupture. Moreover, alanine scanning analysis identified four critical hydrophobic amino acids in the RAM13 domain essential for its cytotoxicity. Consequently, these results suggest that Tat-Ram13 is a tumor-selective, nonapoptotic cell death-inducing agent for treating refractory leukemia and lymphomas with apoptosis resistance.

Developmental Stage and Cellular Context Determine Oncogenic and Molecular Outcomes of Ezh2 Y641F Mutation in Hematopoiesis.

Mutations in the histone methyltransferase EZH2, particularly the Y641 hotspot mutation, have been implicated in hematologic malignancies, yet the effect of timing and cellular context on their oncogenic potential has remained unknown. In this study, we utilized a conditional allele with tissue-specific Cre drivers to investigate the effects of Ezh2 Y641F mutations at various stages of development, with a focus on the hematopoietic system. We found that ubiquitous heterozygous Ezh2 Y641F expression at birth, or conditional expression in hematopoietic or mesenchymal stem cells, led to decreased survival due to hematopoietic defects and bone marrow failure, with no evidence of malignancy. In contrast, Ezh2 Y641F expression in committed B cells drives lymphoma formation, highlighting the lineage-specific oncogenic activity of the mutation. Transcriptomic analysis of B cell progenitors revealed key pathway alterations between Cre models such as altered IL2-Stat5 signaling pathway, differential expression of E2F targets, and altered GTPase pathway expression driven by upregulation of Guanylate Binding Proteins (GBPs) in Mx1-Cre Ezh2 Y641F pro-B cells. We further found that the GBP locus is regulated by Ezh2-mediated H3K27me3, it is associated with poorer survival in Acute Myeloid Leukemia patients and has variable effects on apoptosis in human lymphoma and leukemia cell lines. These findings suggest that the Ezh2 Y641F mutation may alter immune regulatory pathways, cell differentiation and apoptosis, with potential implications for disease progression. Our results highlight the critical role of mutation timing and cellular context in EZH2-driven hematopoietic disease, resulting in distinct downstream changes that shape the oncogenic impact of EZH2.

Evaluation of the antitrypanosomal activity, cytotoxicity and phytochemistry of red Brazilian propolis.

Recently, the growth in the consumption of functional foods with potential nutritional and health benefits revealed rapid progress in phytochemical analysis to assure quality and profile the chemical composition. Bee propolis, a gummy exudate produced in beehives after harvesting from different plant species and showed to contain bioactive secondary metabolites with biological importance. The main goal of the current study is to profile the chemical composition of red propolis samples from the Brazilian stingless bee Tetragonula biroi for the first time using HPLC-UV-ELSD and NMR analysis for assignment of the abundant metabolites' classes as well as extraction and isolation of the major compounds. Column chromatography and size exclusion chromatography were applied for the purification of the major compounds in red Brazilian propolis. Further, testing the antitrypanosomal and cytotoxic activities against Trypanosoma brucei and human leukemia cell lines (U937) was performed. A total of 29 secondary metabolites were identified as two anthocyanins, 6 flavonoids, 8 isoflavonoids, 10 phenolics, two phenolic acids, and one triterpenoid. Two phenolic compounds were purified and identified using 1D and 2D NMR analysis along with MS analysis as liquiritigenin and calycosin. Red Brazilian propolis FB-3 fraction showed the highest inhibitory activity against T. brucei at 1.6 μg/ml, compared to 12.4 μg/ml of the crude extract. The isolated compounds showed moderate activity with an MIC of 8.5 μg/ml for liquiritigenin and 8.7 μg/ml for calycosin. Moreover, FB-3 fraction and calycosin were showed the potent cytotoxic effect with IC50 = 45.1 and 35.8μg/ml, respectively compared to IC50 = 29.5 μg/ml of the standard diminazen. Hence, red Brazilian propolis is rich source of polyphenols with myriad biological importance. Propolis fractions and purified compounds showed moderate antiprotozoal activity and potent cytotoxic activity against human leukemia cell lines.

The synthesis and antileukemic activity of 5-substituted thiazolyl urea derivatives

A series of novel 5-substituted thiazolyl urea derivatives were synthesized and evaluated for their efficacy as antileukemic agents against two human leukemic cell lines (THP-1 and MV-4-11). Results showed that the activities of the investigated compounds were quite sensitive to the positions and properties of the aromatic substituents. Among these compounds, compound 12k showed the highest activity with IC50 values of 29 ± 0.3 nM for THP-1 cells and 98 ± 10 nM for MV-4-11 cells.

Comparative small molecule screening of primary human acute leukemias, engineered human leukemia and leukemia cell lines

Targeted therapeutics for high-risk cancers remain an unmet medical need. Here we report the results of a large-scale screen of over 11,000 molecules for their ability to inhibit the survival and growth in vitro of human leukemic cells from multiple sources including patient samples, de novo generated human leukemia models, and established human leukemic cell lines. The responses of cells from de novo models were most similar to those of patient samples, both of which showed striking differences from the cell-line responses. Analysis of differences in subtype-specific therapeutic vulnerabilities made possible by the scale of this screen enabled the identification of new specific modulators of apoptosis, while also highlighting the complex polypharmacology of anti-leukemic small molecules such as shikonin. These findings introduce a new platform for uncovering new therapeutic options for high-risk human leukemia, in addition to reinforcing the importance of the test sample choice for effective drug discovery.

Pyroptosis is not likely to play a major role in anthracycline-induced cytotoxicity in H9c2 cardiomyocytes and human cardiac endothelial cells

Abstract Background Although anthracyclines are widely used as effective chemotherapeutic agents, anthracycline-induced cardiotoxicity (AIC) causes significant morbidity and mortality. The precise mechanism of AIC remains elusive, although apoptosis may be involved. Recent research suggests pyroptosis, a programmed lytic cell death pathway, might also contribute to AIC. Although studies have focused on AIC in the myocardium, emerging evidence suggests that AIC may also affect vascular cells. Furthermore, the cytotoxicity of doxorubicin (DOX) or its supposed active metabolite, doxorubicinol (DOXOL), has not been widely investigated in cardiac vascular cells. Purpose This study examines the hypothesis that pyroptosis plays a role in DOX and DOXOL cytotoxicity in H9c2 immortalised rat cardiac myoblasts and two types of human cardiac endothelial cells. Methods In vitro experiments were conducted to ascertain dose-dependent toxicity in H9c2, and cardiac endothelial cells following treatment with DOX or DOXOL. The type of cell death was determined by assessing cell morphology and fluorescent staining with markers of apoptosis (Annexin V) and lytic cell death (Propidium Iodide PI) following DOX or DOXOL treatment in comparison to known inducers of pyroptosis (LPS & Nigericin). Western blot experiments were conducted to study the expression of GSDMD (involved in pyroptosis), and caspase 3 (involved in apoptosis), following DOX or DOXOL. A human monocytic acute myeloid leukaemia cell line (THP-1 cells) was used as a positive control for pyroptosis. Results Dose-dependent cell death following DOX treatment was higher than with DOXOL in both H9c2 and cardiac endothelial cells. DOX caused apoptotic changes in all cell types. In contrast, DOXOL had weaker effects, inducing early apoptosis. No cleavage (activation) of GSDMD or caspase 3 was detected after DOX or DOXOL. Interestingly, in THP-1 cells, DOX induced predominantly apoptotic cell death, whereas DOXOL induced predominantly pyroptotic cell death, assessed both morphologically and by caspase 3 and GSDMD cleavage. Conclusion DOX and DOXOL primarily induce apoptotic cell death in H9c2 cardiomyocytes and cardiac endothelial cells. Interestingly, DOXOL, supposedly the active metabolite, was less cytotoxic than DOX. THP-1 cells undergo apoptotic or pyroptotic cell death with DOX or DOXOL, respectively. In conclusion, anthracycline-induced cytotoxicity is likely to be due to apoptotic injury rather than pyroptosis.

Progesterone decreases viability and up regulates membrane progesterone receptors expression on the human Chronic Myeloid Leukemia cell line

Progesterone (P4) has an important effect (activatory or inhibitory) on cell proliferation. Although there is evidence of the impact of progesterone on sex-linked cancers, it can affect other cancer cells expressing P4 receptors (PRs). We evaluated the expression of membrane P4 receptors (mPRs) and the viability in progesterone-treated K562 cells to inspect the possible effects route of progesterone on this (CML) cancer cell line. K562 cells were exposed to various concentrations of progesterone or no exposure for 48 and 72 h. The percentage of viability and cells that expressed mPRα and mPRβ were evaluated by MTT test and flow cytometry respectively. Progesterone significantly increased the expression of mPRα and especially mPRβ on the surface of K562 cells and significantly decreased their viability (p ≤ 0.05). Progesterone can reduce viability in K562 cells. Our findings showed that progesterone affects its receptor expression on K562 cells. Thus it may influence the performance of K562 cells in addition to its direct effects on these cells (via binding to its receptors).

Investigation of Apoptosis-mediated Cytotoxic Effects of Royal Jelly on HL-60 Cells

In recent years, the use of nontoxic natural products that can be effective on cancer cells as new agents has attracted the attention of scientists in order to reduce the negative side effects of existing cancer drugs and their toxicity to normal cells. Some in vivo and in vitro studies have shown that royal jelly (RJ) inhibits cell proliferation and induces apoptosis. In this research, we aimed to investigate the effects of RJ on proliferative and apoptotic processes in the human acute promyelocytic leukemia cell line (HL-60). The HL60 cell line was treated with different concentrations of RJ for 24, 48, and 72 hours. The half maximum inhibitory concentration (IC50) of RJ was determined using 3-(4.5 dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide test (MTT) and the proliferation activity of HL-60 cells was evaluated. Flow cytometry analysis was performed to measure apoptosis in HL-60 cells. IC50 values for RJ were calculated as 13.98, 6.45, and 2.06 mg/mL for 24, 48, and 72 hours, respectively. Flow cytometry results also showed that RJ had apoptotic effects at the concentrations found. The results showed that RJ treatment significantly induced apoptosis and reduced the proliferation of HL-60 cells. This study shows that RJ can be a complementary treatment against HL-60 acute myeloid leukemia cells due to its anticancer and antiproliferative effects.

High glucose condition aggravates inflammatory response induced by Porphyromonas gingivalis in THP-1 macrophages via autophagy inhibition

BackgroundPorphyromonase gingivalis (P. gingivalis) is a type of bacteria that causes periodontitis, which is strongly correlated with systemic diseases such as diabetes. However, the effect of hyperglycemia on periodontitis are unclear. The present study examined the effects of high glucose levels on the response to P. gingivalis infection.ResultsThe expression of P. gingivalis-induced interleukin-1β (IL-1β) and inflammasomes increased as the glucose concentration increased. High glucose conditions suppressed P. gingivalis–induced autophagy in human acute monocytic leukemia cell line (THP-1) macrophages. Zingerone increased autophagy and alleviated P. gingivalis-induced inflammatory response in THP-1 macrophages under high glucose conditions. In addition, P. gingivalis- induced inflammation in bone marrow-derived macrophages of diabetic mice was higher than in wild-type mice, but a zingerone treatment decreased the levels. Alveolar bone loss due to a P. gingivalis infection was significantly higher in diabetic mice than in wild-type mice.ConclusionsHigh-glucose conditions aggravated the inflammatory response to P. gingivalis infection by suppressing of autophagy, suggesting that autophagy induction could potentially to treat periodontitis in diabetes. Zingerone has potential use as a treatment for periodontal inflammation induced by P. gingivalis in diabetes patients.

BCAT1 contributes to the development of TKI-resistant CML.

Although most of chronic myeloid leukemia (CML) patients can be effectively treated by the tyrosine kinase inhibitors (TKIs), such as Imatinib, TKI-resistance still occurs in approximately 15-17% of cases. Although many studies indicate that branched chain amino acid (BCAA) metabolism may contribute to the TKI resistance in CML, the detailed mechanisms remains largely unknown. The cell proliferation, colony formation and in vivo transplantation were used to determined the functions of BCAT1 in leukemogenesis. Quantitative real-time PCR (RT-PCR), western blotting, RNA sequencing, BCAA stimulation in vitro were applied to characterize the underlying molecular mechanism that control the leukemogenic activity of BCAT1-knockdown cells. In this report, we revealed that branched chain amino acid transaminase 1 (BCAT1) is highly enriched in both mouse and human TKI-resistant CML cells. Leukemia was almost completely abrogated upon BCAT1 knockdown during transplantation in a BCR-ABLT315I-induced murine TKI-resistant CML model. Moreover, knockdown of BCAT1 led to a dramatic decrease in the proliferation of TKI-resistant human leukemia cell lines. BCAA/BCAT1 signaling enhanced the phosphorylation of CREB, which is required for maintenance of TKI-resistant CML cells. Importantly, blockade of BCAA/BCAT1 signaling efficiently inhibited leukemogenesis both in vivo and in vitro. These findings demonstrate the role of BCAA/BCAT1 signaling in cancer development and suggest that targeting BCAA/BCAT1 signaling is a potential strategy for interfering with TKI-resistant CML.

Targeting USP14/UCHL5: A Breakthrough Approach to Overcoming Treatment-Resistant FLT3-ITD-Positive AML.

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations in acute myeloid leukemia (AML) are associated with poor prognosis and therapy resistance. This study aimed to demonstrate that inhibiting the deubiquitinating enzymes ubiquitin-specific peptidase 14 (USP14) and ubiquitin C-terminal hydrolase L5 (UCHL5) (USP14/UCHL5) with b-AP15 or the organogold compound auranofin (AUR) induces apoptosis in the ITD-transformed human leukemia cell line MV4-11 and mononuclear leukocytes derived from patients with FLT3-ITD-positive AML. This study included patients diagnosed with AML at Tokyo Medical and Dental University Hospital between January 2018 and July 2024. Both treatments blocked downstream FLT3 pathway events, with the effects potentiated by USP14 knockdown. Both treatments inhibited FLT3 deubiquitination via K48 and disrupted translation initiation via 4EBP1, a downstream FLT3 target. FLT3 was downregulated in the leukemic cells, with the associated activation of stress-related MAP kinase pathways and increased NF-E2-related factor 2. Furthermore, the overexpression of B-cell lymphoma-extra-large and myeloid cell leukemia-1 prevented the cell death caused by b-AP15 and AUR. These results suggest that inhibiting USP14/UCHL5, which involves multiple regulatory mechanisms, is a promising target for novel therapies for treatment-resistant FLT3-ITD-positive AML.

Novel alloxazine analogues: design, synthesis, and antitumour efficacy enhanced by kinase screening, molecular docking, and ADME studies

This study describes the development of novel alloxazine analogues as potent antitumor agents with enhanced selectivity for tumour cells. Twenty-nine out of 45 newly compounds were investigated in vitro for their growth inhibitory activities, against two human tumour cell lines, namely, the human T-cell acute lymphoblastoid leukaemia cell line (CCRF-HSB-2) and human oral epidermoid carcinoma cell line (KB), and the antitumor agent “Ara-C” was used as a positive reference in this investigation. Compounds 9e and 10J were the highest among their analogues, against both tumour cell lines (CCRF-HSB-2 and KB). Correlation analyses demonstrated a strong relationship between the IC50 values and AutoDock binding free energy or calculated inhibition (Ki ). The study delves into structure–activity relationships (SARs) through advanced modelling tools integrated with structure-based drug design (SBDD) using GOLD 5.2.2, AutoDock 4.2, and Accelrys Discovery Studio 3.5. Physicochemical properties, pharmacokinetics, drug-likeness, and toxicity predictions of the most potent alloxazine derivatives were conducted using ProTox-II and Swiss ADME for effective antitumor agents with improved selectivity.

Selective degradation of mutant FMS-like tyrosine kinase-3 requires BIM-dependent depletion of heat shock proteins

Internal tandem duplications in the FMS-like tyrosine kinase-3 (FLT3-ITD) are common mutations in acute myeloid leukemia (AML). Proteolysis-targeting chimeras (PROTACs) that induce proteasomal degradation of mutated FLT3 emerge as innovative pharmacological approach. Molecular mechanisms that control targeted proteolysis beyond the ubiquitin-proteasome-system are undefined and PROTACs are the only known type of FLT3 degraders. We report that the von-Hippel-Lindau ubiquitin-ligase based FLT3 PROTAC MA49 (melotinib-49) and the FLT3 hydrophobic tagging molecule MA50 (halotinib-50) reduce endoplasmic reticulum-associated, oncogenic FLT3-ITD but spare FLT3. Nanomolar doses of MA49 and MA50 induce apoptosis of human leukemic cell lines and primary AML blasts with FLT3-ITD (p < 0.05-0.0001), but not of primary hematopoietic stem cells and differentiated immune cells, FLT3 wild-type cells, retinal cells, and c-KIT-dependent cells. In vivo activity of MA49 against FLT3-ITD-positive leukemia cells is verified in a Danio rerio model. The degrader-induced loss of FLT3-ITD involves the pro-apoptotic BH3-only protein BIM and a previously unidentified degrader-induced depletion of protein-folding chaperones. The expression levels of HSP90 and HSP110 correlate with reduced AML patient survival (p < 0.1) and HSP90, HSP110, and BIM are linked to the expression of FLT3 in primary AML cells (p < 0.01). HSP90 suppresses degrader-induced FLT3-ITD elimination and thereby establishes a mechanistically defined feed-back circuit.

Phytochemical profiling and characterization of flavonoid derivatives from propolis sample and investigation of cytotoxic and antiprotozoal activities

Recently, the growth of consumer demand for functional foods with potential nutritional and health benefits led to rapid growth of analytical tools for profiling of bioactive metabolites and assure quality. Bee propolis is one of the most important bee products owing to its myriad health value. As a gummy exudate produced in beehives after harvesting from different plant species, bee propolis contains bioactive secondary metabolites. The current study aims to profiling the chemical composition of propolis samples from Nigeria using HPLC–UV-ELSD and with the aid of NMR-based analysis for assignment of metabolites classes abundant in Nigerian propolis. Red Nigerian propolis samples were subjected to phytochemical analysis using HPLC–UV-ELSD and NMR. Further chromatographic separation of promising fractions was performed by column chromatography and size exclusion chromatography. Screening of the antitrypanosomal and cytotoxic activities against Trypanosoma brucei and human leukemia cell lines (U937), respectively, was performed. The performance of LC–MS permitted identification of the different components from which 13 compound were identified and allowed combination of fractions to afford 9 fractions from which two isoflavonoids were isolated and identified using 1D and 2D NMR analysis with MS as isosativan and Medicarpin. Red Nigerian propolis crude extract showed the highest inhibitory activity at 6.5 µg/ml compared to moderate activity for the isolated compounds with MIC of 7.6 µg/ml and 12.1 µg/ml for medicarpin and isosativan, respectively. Moreover, the fraction RN-6 from the total extract showed the potent cytotoxic effect with IC50 = 26.5 µg/ml compared to standard diminazen which showed IC50 = 29.5 µg/ml.