Human Acute Myeloid Leukemia Cells Research Articles

Platycodin D induces apoptotic cell death through PI3K/AKT and MAPK/ERK pathways and synergizes with venetoclax in acute myeloid leukemia

Acute myeloid leukemia (AML) is a highly heterogeneous and rapidly progressive hematopoietic neoplasm characterized by frequent relapses and variable prognoses. The development of new treatment options, therefore, is of crucial importance. Platycodin D (PD) is a triterpenoid saponin, extracted from the roots of the traditional Chinese herbal medicine Platycodon grandiflorum (Jacq.) A. DC., which has been reported to exhibit therapeutic potential against a broad range of cancers. Although the effects of PD on AML remain unclear, in the present study, we observed a concentration-dependent reduction in the viability of multiple human AML cell lines in response to treatment with PD. In addition to triggering mitochondria-dependent apoptosis via the upregulation of BAK and BIM, treatment with PD also induced cell cycle arrest at the G0/G1 phase. Western blot analyses revealed marked suppression of the phosphorylation of protein kinase B (AKT), glycogen synthase kinase-3β, ribosomal protein S6, and extracellular signal-regulated kinase (ERK) by PD, in turn implying the participation of the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/ERK pathways. Pre-incubation with LY294002, MK2206, AR-A014418, or U0126 was consistently found to significantly aggravate PD-induced inhibition of viability. Additionally, PD combined with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax elicited synergistically enhanced cytotoxic effects. The anti-leukemic activity of PD was further validated using primary samples from de novo AML patients. Given the results of the present study, PD may be a potent therapeutic candidate for the treatment of AML.

Casein Kinase 2 (CK2): A Possible Therapeutic Target in Acute Myeloid Leukemia.

The protein kinase CK2 (also known as casein kinase 2) is one of the main contributors to the human phosphoproteome. It is regarded as a possible therapeutic strategy in several malignant diseases, including acute myeloid leukemia (AML), which is an aggressive bone marrow malignancy. CK2 is an important regulator of intracellular signaling in AML cells, especially PI3K-Akt, Jak-Stat, NFκB, Wnt, and DNA repair signaling. High CK2 levels in AML cells at the first time of diagnosis are associated with decreased survival (i.e., increased risk of chemoresistant leukemia relapse) for patients receiving intensive and potentially curative antileukemic therapy. However, it is not known whether these high CK2 levels can be used as an independent prognostic biomarker because this has not been investigated in multivariate analyses. Several CK2 inhibitors have been developed, but CX-4945/silmitasertib is best characterized. This drug has antiproliferative and proapoptotic effects in primary human AML cells. The preliminary results from studies of silmitasertib in the treatment of other malignancies suggest that gastrointestinal and bone marrow toxicities are relatively common. However, clinical AML studies are not available. Taken together, the available experimental and clinical evidence suggests that the possible use of CK2 inhibition in the treatment of AML should be further investigated.

The ribosomal protein S6 kinase alpha-1 (RPS6KA1) induces resistance to venetoclax/azacitidine in acute myeloid leukemia

Venetoclax/azacitidine combination therapy is effective in acute myeloid leukemia (AML) and tolerable for older, multimorbid patients. Despite promising response rates, many patients do not achieve sustained remission or are upfront refractory. Identification of resistance mechanisms and additional therapeutic targets represent unmet clinical needs. By using a genome-wide CRISPR/Cas9 library screen targeting 18,053 protein- coding genes in a human AML cell line, various genes conferring resistance to combined venetoclax/azacitidine treatment were identified. The ribosomal protein S6 kinase A1 (RPS6KA1) was among the most significantly depleted sgRNA-genes in venetoclax/azacitidine- treated AML cells. Addition of the RPS6KA1 inhibitor BI-D1870 to venetoclax/azacitidine decreased proliferation and colony forming potential compared to venetoclax/azacitidine alone. Furthermore, BI-D1870 was able to completely restore the sensitivity of OCI-AML2 cells with acquired resistance to venetoclax/azacitidine. Analysis of cell surface markers revealed that RPS6KA1 inhibition efficiently targeted monocytic blast subclones as a potential source of relapse upon venetoclax/azacitidine treatment. Taken together, our results suggest RPS6KA1 as mediator of resistance towards venetoclax/azacitidine and additional RPS6KA1 inhibition as strategy to prevent or overcome resistance.

FOXP1 regulates oxidative stress, SIRT1 expression, and resistance to chemotherapies in acute myeloid leukemia cells.

Transcription factor Forkhead box P1 (FOXP1) belongs to the same protein family as the FOXOs that are well-known regulators of murine hematopoietic stem progenitor cell (HSPC) maintenance by dampening oxidative stress. FOXP1 and FOXOs can play opposite or similar roles depending on cell context; they can cross-regulate each other's expression. In a previous study, we have shown that FOXP1 contributes to normal human HSP and acute myeloid leukemia (AML) cell growth. Here we investigated the role of FOXP1 in HSPCs and AML cell oxidative stress defense in human context. FOXP1 expression level was associated with inferior survival outcome of cytogenetically normal (CN) AML patients. FOXP1 knockdown enhanced superoxide anion levels of human committed CD34+CD38+ but not stem cell-enriched CD34+CD38- HSPCs, and AML cells in vitro. It triggered enhanced NRF2 activity and increased cell oxidative stress. FOXP1 had no impact on FOXO1/3/4 expression in these cells; genetic and pharmacological inhibition of FOXOs did not change superoxide anion levels of human HSPCs and AML cells. Also, FOXP1 antioxidant activity was independent of superoxide dismutase (SOD)1-2 or catalase expression changes. Instead, FOXP1 upregulated expression of the stress sensor SIRT1 by stabilizing SIRT1 protein. FOXP1 loss sensitized AML cells to chemotherapy. Altogether, this study identified FOXP1 as a new safeguard against myeloid progenitor oxidative stress, which works independently of FOXOs but through SIRT1, and contributes to AML chemoresistance. It proposes FOXP1 expression/activity as a promising target to overcome drug-resistance of AML HSPCs.

Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

Relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia-initiating stem cells (LICs) that are typically not targeted by most existing therapies. Using a murine AML model, human AML cell lines, and patient samples, we show that AML LICs are sensitive to endogenous and exogenous cyclopentenone prostaglandin-J (CyPG), Δ12-PGJ2, and 15d-PGJ2, which are increased upon dietary selenium supplementation via the cyclooxygenase-hematopoietic PGD synthase pathway. CyPGs are endogenous ligands for peroxisome proliferator-activated receptor gamma and GPR44 (CRTH2; PTGDR2). Deletion of GPR44 in a mouse model of AML exacerbated the disease suggesting that GPR44 activation mediates selenium-mediated apoptosis of LICs. Transcriptomic analysis of GPR44-/- LICs indicated that GPR44 activation by CyPGs suppressed KRAS-mediated MAPK and PI3K/AKT/mTOR signaling pathways, to enhance apoptosis. Our studies show the role of GPR44, providing mechanistic underpinnings of the chemopreventive and chemotherapeutic properties of selenium and CyPGs in AML.

Phase 1 study of ziftomenib (KO-539) in combination with venetoclax or venetoclax/azacitidine or standard induction cytarabine/daunorubicin (7+3) chemotherapy for the treatment of patients with acute myeloid leukemia.

TPS7079 Background: Relapsed or refractory (R/R) acute myeloid leukemia (AML) with nucleophosmin 1-mutations ( NPM1-m) or KMT2A-rearrangment ( KMT2A-r) represent a high unmet need. Ziftomenib is a potent and selective inhibitor that targets the menin-mixed-lineage leukemia (MLL) lysine[K]-specific methyltransferase 2A ( KMT2A) interaction driving leukemogenesis in these subtypes. An ongoing Phase 1/2 study (KO-MEN 001) has demonstrated meaningful clinical activity and tolerability with ziftomenib monotherapy in R/R AML. In preclinical testing, ziftomenib combined with venetoclax induced synergistic lethality in KMT2A-r and NPM1-m human AML cell lines and patient-derived cells. Moreover, treatment with ziftomenib plus venetoclax/azacitidine has induced prolonged durable remissions in mice with KMT2A-r AML xenografts. Therefore, administration of ziftomenib in combination with standard-of-care (SOC) therapies may provide additional clinical benefit for patients with newly diagnosed or R/R NPM1-m and KMT2A-r menin-dependent AML. Methods: KOMET-007 (KO-MEN-007, NCT# ) is a 2-part dose escalation (Phase 1a) and expansion (Phase 1b) study to evaluate the safety, tolerability, and preliminary antileukemic activity of ziftomenib in combination with non-intensive chemotherapy (NIC) and intensive chemotherapy (IC) in patients with NPM1-m and KMT2A-r AML. During Phase 1a, the ziftomenib dose will be escalated with standard doses of either venetoclax and azacitidine (zifto/ven/aza) or cytarabine and daunorubicin (zifto/7+3) in defined genetic cohorts ( NPM1-m and KMT2A-r) using a rule-based approach (n = 6 per cohort/dose level) to select ziftomenib doses for expansion/validation in Phase 1b. The Phase 1b portion will also evaluate zifto/ven/aza in newly diagnosed AML ( KMT2A-r and NPM1-m) and zifto/ven in R/R AML ( NPM1-m only). The overall primary objectives are to establish the minimum biologically effective, dose limiting toxicities, and recommended Phase 2 dose of ziftomenib combined with NIC and IC backbones in each genetically defined cohort. Additional secondary endpoints include preliminary antileukemic efficacy (including MRD-negative responses and number of patients able to transition to subsequent allogeneic stem cell transplant), pharmacokinetic, and pharmacodynamic data. Exploratory end points include biomarkers for efficacy and resistance, pharmacodynamic biomarkers and activity in isolated myeloid sarcoma. Clinical trial information: NCT04067336 .

KMT2D Deficiency Promotes Myeloid Leukemias which Is Vulnerable to Ribosome Biogenesis Inhibition.

KMT2C and KMT2D are the most frequently mutated epigenetic genes in human cancers. While KMT2C is identified as a tumor suppressor in acute myeloid leukemia (AML), the role of KMT2D remains unclear in this disease, though its loss promotes B cell lymphoma and various solid cancers. Here, it is reported that KMT2D is downregulated or mutated in AML and its deficiency, through shRNA knockdown or CRISPR/Cas9 editing, accelerates leukemogenesis in mice. Hematopoietic stem and progenitor cells and AML cells with Kmt2d loss have significantly enhanced ribosome biogenesis and consistently, enlarged nucleolus, increased rRNA and protein synthesis rates. Mechanistically, it is found that KMT2D deficiency leads to the activation of the mTOR pathway in both mouse and human AML cells. Kmt2d directly regulates the expression of Ddit4, a negative regulator of the mTOR pathway. Consistent with the abnormal ribosome biogenesis, it is shown that CX-5461, an inhibitor of RNA polymerase I, significantly restrains the growth of AML with Kmt2d loss in vivo and extends the survival of leukemic mice. These studies validate KMT2D as a de facto tumor suppressor in AML and reveal an unprecedented vulnerability to ribosome biogenesis inhibition.

Targeting human mitochondrial NAD(P)+-dependent malic enzyme (ME2) impairs energy metabolism and redox state and exhibits antileukemic activity in acute myeloid leukemia

Acute myeloid leukemia (AML) is a fast-growing and highly fatal blood cancer, and recent research has shown that targeting metabolism may be a promising therapeutic approach for treating AML. One promising target is the human mitochondrial NAD(P)+-dependent malic enzyme (ME2), which is involved in the production of pyruvate and NAD(P)H and the regulation of the NAD+/NADH redox balance. Inhibition of ME2 via silencing ME2 or utilizing its allosteric inhibitor disodium embonate (Na2EA) causes a decrease in pyruvate and NADH, leading to a decrease in producing ATP via cellular respiration and oxidative phosphorylation. ME2 inhibition also decreases NADPH levels, resulting in an increase in reactive oxygen species (ROS) and oxidative stress, which ultimately leads to cellular apoptosis. Additionally, ME2 inhibition reduces pyruvate metabolism and the biosynthetic pathway. ME2 silencing inhibits the growth of xenotransplanted human AML cells, and the allosteric ME2 inhibitor Na2EA demonstrates antileukemic activity against immune-deficient mice with disseminated AML. Both of these effects are a result of impaired energy metabolism in mitochondria. These findings suggest that the targeting ME2 may be an effective strategy for treating AML. Overall, ME2 plays an essential role in energy metabolism of AML cells, and its inhibition may offer a promising approach for AML treatment.

Abstract 525: Targeting interleukin-1 receptor-associated kinase 1/4 in acute myeloid leukemia stem cells

Abstract Therapies for acute myeloid leukemia (AML) patients are suboptimal due mainly to relapse of disease after treatment. Leukemia stem cell (LSC) populations maintain the disease and serve as a reservoir for relapse. Therefore, treatments that can eradicate LSCs will improve the outcomes of patients with AML. Our preliminary data have shown that the interleukin-1 (IL-1) pathway is upregulated following treatment and disease progression in AML and plays an important role in LSCs in vitro and in vivo. To target IL-1 signaling, we disclose the structure-activity relationship guided development of UR241-2, a novel small molecule compound of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4), which are critical downstream factors in IL-1/Toll-Like Receptor (TLR) signaling. UR241-2, at single dose (5 nM) screening exhibited high target selectivity; other than IRAK1/4 only 9 kinases in a panel of 682 kinases were inhibited by >25% activity. The NanoBRET assay using HEK293 cells transfected with targeted kinase vectors showed notably selective target engagement of UR241-2 against IRAK4 (IC50 = 6.07 nM). By using the in vitro NF-kB reporter assay and immunoblotting analysis, we identified that UR241-2 robustly inhibits IL-1/TLR signaling in AML cells including the activation of NF-κB and the phosphorylation of P65 and P38, following IL-1 stimulation. Next, we treated murine MLL-AF9 AML cells and human MDSL cells with UR241-2 and two IRAK4 inhibitors (PF-06650833 and CA-4948) that are currently in clinical or preclinical studies. We found that UR241-2 significantly inhibits colony-forming ability in AML and myelodysplastic syndromes, with similar or better efficacy compared to PF-06650833 or CA-4948. We also determined that UR241-2 suppresses LSC clonogenicity in primary human AML cells at diagnosis and in relapse while minimally impacting normal hematopoietic stem and progenitor cell function. Last, we investigated the impacts of UR241-2 on LSC engrafted ability in vivo. We treated murine MLL-AF9 cells with UR241-2 and PF-06650833 ex vivo for three days and transplanted them into recipient mice. After four weeks, we observed that UR241-2 significantly suppresses the engraftment levels of AML cells in bone marrow. It also decreases the WBC in peripheral blood and increases the RBC, HGB and PLT counts compared to the vehicle control group. Taken together, our findings demonstrated that targeting IRAK1/4 using the novel compound UR241-2 is able to impair LSC clonogenicity and engraftment capability in AML following treatment and disease progression. This provides a potential therapeutic strategy to eradicate the LSC population and improve the outcome of AML patients. Citation Format: Tzu-Chieh Ho, Rakesh K. Singh, Hiroki Kawano, Mark W. LaMere, Jian Wang, Paula M. Vertino, Nikolay V. Dokholyan, Laura M. Calvi, Craig T. Jordan, Michael W. Becker. Targeting interleukin-1 receptor-associated kinase 1/4 in acute myeloid leukemia stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 525.

Tespa1 facilitates hematopoietic and leukemic stem cell maintenance by restricting c-Myc degradation

Hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) have robust self-renewal potential, which is responsible for sustaining normal and malignant hematopoiesis, respectively. Although considerable efforts have been made to explore the regulation of HSC and LSC maintenance, the underlying molecular mechanism remains obscure. Here, we observe that the expression of thymocyte-expressed, positive selection-associated 1 (Tespa1) is markedly increased in HSCs after stresses exposure. Of note, deletion of Tespa1 results in short-term expansion but long-term exhaustion of HSCs in mice under stress conditions due to impaired quiescence. Mechanistically, Tespa1 can interact with CSN subunit 6 (CSN6), a subunit of COP9 signalosome, to prevent ubiquitination-mediated degradation of c-Myc protein in HSCs. As a consequence, forcing c-Myc expression improves the functional defect of Tespa1-null HSCs. On the other hand, Tespa1 is identified to be highly enriched in human acute myeloid leukemia (AML) cells and is essential for AML cell growth. Furthermore, using MLL-AF9-induced AML model, we find that Tespa1 deficiency suppresses leukemogenesis and LSC maintenance. In summary, our findings reveal the important role of Tespa1 in promoting HSC and LSC maintenance and therefore provide new insights on the feasibility of hematopoietic regeneration and AML treatment.

Enzymatic Synthesis of 2-Chloropurine Arabinonucleosides with Chiral Amino Acid Amides at the C6 Position and an Evaluation of Antiproliferative Activity In Vitro

A number of purine arabinosides containing chiral amino acid amides at the C6 position of the purine were synthesized using a transglycosylation reaction with recombinant E. coli nucleoside phosphorylases. Arsenolysis of 2-chloropurine ribosides with chiral amino acid amides at C6 was used for the enzymatic synthesis, and the reaction equilibrium shifted towards the synthesis of arabinonucleosides. The synthesized nucleosides were shown to be resistant to the action of E. coli adenosine deaminase. The antiproliferative activity of the synthesized nucleosides was studied on human acute myeloid leukemia cell line U937. Among all the compounds, the serine derivative exhibited an activity level (IC50 = 16 μM) close to that of Nelarabine (IC50 = 3 μM) and was evaluated as active.

Cytarabine and dexamethasone-PAMAM dendrimer di-conjugate sensitizes human acute myeloid leukemia cells to apoptotic cell death

Polyamidoamine (PAMAM) dendrimers are widely used as a part of drug delivery systems (DDS) to improve anticancer drug bioavailability. In the present study, monophosphates of nucleosides, anti-nucleosides (cytarabine, C and fludarabine, F) and synthetic glucocorticoid dexamethasone (D) were attached to the third generation (G3) PAMAM dendrimer via phosphamide pH-sensitive linker. Conjugates were found to be stable at neutral pH, whereas acid-triggered hydrolysis of phosphamide bond was observed at pH 5. The anticancer action of selected biotinylated mono- and di-conjugates, namely CP-, FP-, DP-CP- and DP-FP-PAMAM dendrimers was then compared to cytotoxic activity of free drugs using four cellular models of leukemia in vitro, i.e., K-562, LAMA-84, THP-1 and HL-60 cells. 10 nM DP-CP-conjugate lowered metabolic activity, caused G0/G1 cell cycle arrest and induced apoptotic cell death in HL-60 cells compared to non-cytotoxic and non-cytostatic action of free drugs when used at the same concentration. 10 nM CP-, DP-FP- and FP-conjugates also promoted a decrease in metabolic activity in K-562, LAMA-84, and THP-1 and HL-60 cells, respectively, however, without a sign of cytotoxicity. In conclusion, we show that the anticancer potential of CP and FP in leukemia cells may be enhanced by the conjugation with PAMAM dendrimers, especially in the presence of DP in selected experimental settings.

Synthetic lethal targeting of TET2-mutant haematopoietic stem and progenitor cells by XPO1 inhibitors.

TET2 inactivating mutations serve as initiating genetic lesions in the transformation of haematopoietic stem and progenitor cells (HSPCs). In this study, we analysed known drugs in zebrafish embryos for their ability to selectively kill tet2-mutant HSPCs in vivo. We found that the exportin 1 (XPO1) inhibitors, selinexor and eltanexor, selectively kill tet2-mutant HSPCs. In serial replating colony assays, these small molecules were selectively active in killing murine Tet2-deficient Lineage-, Sca1+, Kit+ (LSK) cells, and also TET2-inactivated human acute myeloid leukaemia (AML) cells. Selective killing of TET2-mutant HSPCs and human AML cells by these inhibitors was due to increased levels of apoptosis, without evidence of DNA damage based on increased γH2AX expression. The finding that TET2 loss renders HSPCs and AML cells selectively susceptible to cell death induced by XPO1 inhibitors provides preclinical evidence of the selective activity of these drugs, justifying further clinical studies of these small molecules for the treatment of TET2-mutant haematopoietic malignancies, and to suppress clonal expansion in age-related TET2-mutant clonal haematopoiesis.

MiR 26 targeting methyltetrahydrofolate reductase inhibits the proliferation of acute myeloid leukemia cells via JAK/STAT pathway

Purpose: To investigate the effect of microribonucleic acid (miR)-26 targeting methyltetrahydrofolate reductase (MTHFR) on cell proliferation, cycle, and apoptosis in acute myeloid leukemia (AML).Methods: The expressions of miR-26 in three types of human AML cell lines (HL-60, Kasumi-1, and KG-1a) and normal myeloid cell line (HS-5) were determined via quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR), while the effect of the over-expression of miR-26 on the proliferation, cell cycle and apoptosis of AML cells was evaluated using cell counting kit-8 (CCK-8) assay and flow cytometry. The potential target for miR-26 was predicted using public miRNA database TargetScan, and whether miR-26 binds to the predicted target was determined using a dual-luciferase reporter assay. Western blotting was performed to determine the effect of miR-26 on Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway-associated proteins.Results: Expression level of miR-26 was significantly lower in all AML cell lines than in HS-5 cells (p < 0.05). Overexpression of miR-26 inhibited the proliferation of KG-1a cells, reduced the percentage of cells in S phase, increased those in G0/G1 phase, and enhanced apoptosis of KG-1a cells (p < 0.05). After overexpression of miR-26, protein expression levels of phosphorylated (p)-JAK and p-STAT were down-regulated, while those of JAK and STAT did not change significantly.Conclusion: Expression of miR-26 is down-regulated in AML, while MiR-26 targeting of MTHFR induces apoptosis and cycle arrest of AML cells through the JAK/STAT pathway, thus inhibiting AML cell proliferation in vitro.

Inhibition of Ferulic Acid on U937 Cell Growth and Its Related Mechanism

To investigate the effects of ferulic acid (FA) on proliferation, apoptosis and Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway of human acute myeloid leukemia (AML) U937 cells. Different concentrations of FA (0、10、25、50、100、200 μmol/L) was used to treat human AML cell lines Kasumi-1, HL-60, and U937 cells respectively for 24 h. The cell survival rate was detected by cell counting kit-8 method, and the 50% inhibitory concentration (IC50) of each cell line was calculated. The U937 cells were divided into control group, FA group (50 μmol/L FA), FA+pcDNA group (50 μmol/L FA+transfected with empty plasmid), FA+pcDNA-TLR4 group (50 μmol/L FA+transfected with TLR4 overexpression plasmid). Flow cytometry was used to detect U937 cell cycle and cell apoptosis; plate clone formation test was used to detect U937 cell clone formation ability; fluorescence quantitative PCR was used to detect the TLR4, NF-κB p65 mRNA levels in U937 cells; Western blot was used to detect the expression levels of CyclinD1, CyclinE, Bcl-2 related X protein (Bax), B cell lymphoma-2 (Bcl-2), Caspase-3, TLR4, and NF-κB p65 protein in U937 cells. With the increase of FA concentration, the survival rates of Kasumi-1, HL-60 and U937 cells gradually decreased(r=-0.919, r=-0.909, r=-0.900), the IC50 of U937 cells was 50.25±2.23 μmol/L. Compared with the control group, after drug treatment of U937 cells, the ratio of G0/G1 phase cells, apoptosis rate, expression levels of Bax and Caspase-3 proteins in FA group were significantly increased (P<0.05), the number of cell clones, the ratios of S phase and G2/M phase cells, expression levels of CyclinD1, CyclinE and Bcl-2 proteins, and TLR4, NF-κB p65 mRNA and protein were significantly decreased (P<0.05); compared with FA group and FA+pcDNA group, the ratio of G0/G1 phase cells, apoptosis rate, expression levels of Bax and Caspase-3 proteins in FA+pcDNA-TLR4 group were significantly decreased (P<0.05), the number of cell clones, the ratios of S phase and G2/M phase cells, expression levels of expression levels of CyclinD1, CyclinE and Bcl-2 proteins, and TLR4, NF-κB p65 mRNA and proteins were significantly increased (P<0.05). FA inhibits U937 cell proliferation and promotes cell apoptosis by inhibiting the TLR4/NF-κB signaling pathway.

Dipeptidylpeptidase 4 promotes survival and stemness of acute myeloid leukemia stem cells.

Leukemic-stem-cell-specific targeting may improve the survival of patients with acute myeloid leukemia (AML) by avoiding the ablative effects of standard regimens on normal hematopoiesis. Herein, we perform an unbiased screening of compounds targeting cell surface proteins and identify clinically used DPP4 inhibitors as strong suppressors of AML development in both murine AML models and primary human AML cells xenograft model. We find in retrovirus-induced AML mouse models that DPP4-deficient AML cell-transplanted mice exhibit delay and reversal of AML development, whereas deletion of DPP4 has no significant effect on normal hematopoiesis. DPP4 activates and sustains survival of AML stem cells that are critical for AML development in both human and animal models via binding with Src kinase and activation of nuclear factor κB (NF-κB) signaling. Thus, inhibition of DPP4 is a potential therapeutic strategy against AML development through suppression of survival and stemness of AML cells.

Novel indole retinoid derivative induces apoptosis and cell cycle arrest and modulates AKT and ERK signaling in HL-60 cells.

Chemotherapy with targeted drugs is the first line therapy option for acute and chronic myeloid leukemia. However, hematopoietic stem cell transplantation may be used in high-risk patients or patients with failed responses to chemo drugs. Discovery and development of more effective new agents with lower side effects is the main aim of leukemia treatment. In this study, a novel retinoid compound with tetrahydronaphthalene ring was synthesized and evaluated for anticancer activity in human chronic and acute myeloid leukemia cell lines K562 and HL-60. Novel N-(1H-indol-1-yl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carboxamide was synthesized based on molecular hybridization of the two different bioactive structures retinoid head and indole. The effects of the synthesized carboxamide compound, which was referred to as compound 5, were determined in K562 chronic myeloid leukemia and HL-60 acute myeloid leukemia cell lines and L929 fibroblast cell line, which served as a control. Colorimetric MTT and caspase3 activity tests, flow cytometry, western blot, and microscopic examinations were used to evaluate biological activity. Compound 5 more effectively induced cell death in HL60 cells in comparison to K562 cells and L929 fibroblast cells. Therefore, further mechanism of cell death was investigated in HL60 cell line. It was found that compound 5 induced remarkable cytotoxicity, caspase3 activation, and PARP fragmentation in HL60 cells. Flow cytometric staining showed that the percentage of cells arrested in G0/G1 was also increased with compound 5 treatment. Important modulator proteins of cell proliferation p-ERK, p-AKT, and p-m-TOR were also found to be inhibited with compound 5 treatment. Collectively, our results reveal compound 5, which is a novel indole retinoid compound as a potential active agent for the treatment of acute promyelocytic leukemia.

Discovery of 4-(4-aminophenyl)-6-phenylisoxazolo[3,4-b]pyridine-3-amine derivatives as novel FLT3 covalent inhibitors for the intervention of acute myeloid leukemia.

Small molecule covalent drugs have proved to be desirable therapies especially on drug resistance related to point mutations. Secondary mutations of FLT3 have become the main mechanism of FLT3 inhibitors resistance which further causes the failure of treatment. Herein, a series of 4-(4-aminophenyl)-6-phenylisoxazolo[3,4-b]pyridine-3-amine covalent derivatives were synthesized and optimized to overcome the common secondary resistance mutations of FLT3. Among these derivatives, compound F15 displayed potent inhibition activities against FLT3 (IC50 = 123 nM) and FLT3-internal tandem duplication (ITD) by 80% and 26.06%, respectively, at the concentration of 1 μM. Besides, F15 exhibited potent activity against FLT3-dependent human acute myeloid leukemia (AML) cell lines MOLM-13 (IC50 = 253 nM) and MV4-11 (IC50 = 91 nM), as well as BaF3 cells with variety of secondary mutations. Furthermore, cellular mechanism assays indicated that F15 inhibited phosphorylation of FLT3 and its downstream signaling factors. Notably, F15 could be considered for further development as potential drug candidate to treat AML.

The histone demethylase KDM5C functions as a tumor suppressor in AML by repression of bivalently marked immature genes

Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.

Effects of olive oil-based propolis, caffeic acid phenethyl ester, and methylprednisolone on differentiation of human acute myeloid leukemia cells

Effects of olive oil-based propolis, caffeic acid phenethyl ester, and methylprednisolone on differentiation of human acute myeloid leukemia cells