KG1a Cells Research Articles

Salvia Miltiorrhiza Injection Inhibited the Proliferation of AML Cells by Inducing Apoptosis through the p38MAPK Pathway.

The purpose of this study was to explore the antitumor effect and mechanism of Salvia miltiorrhiza injection (SMI) on acute myeloid leukemia (AML) cells in vitro and in vivo. Bioinformatics was used to detect c-Myc mRNA expression in AML patients in the Oncomine database. qRT‒PCR and western blotting were used to detect the mRNA and protein expression of c-Myc and HOXA5 in clinical samples. Different concentrations (6.25, 12.5, 25, 50 and 100 μg/mL) of SMI were added to KG1a and HL60 cells for 24, 48 and 72 h to determine the IC50 value of SMI. A CCK-8 assay was used to detect the effects of different concentrations of SMI and different treatment times on the proliferation of KG1a and HL60 cells. The indicated concentrations of SMI and SB203580 were used to treat KG1a and HL60 cells. The cell cycle distribution was determined by flow cytometry. The percentage of apoptotic cells was detected by Hoechst 33258 staining and flow cytometry. qRT‒PCR was performed to detect the mRNA expression of p38, c-Myc and HOXA5 in KG1a and HL60 cells. Western blotting was used to detect the protein expression of p38, p-p38, c-Myc, HOXA5, cCaspase 3 and cPARP in KG1a and HL60 cells. AutoDock software was used to analyze the molecular docking of the three main active components of SMI with c-Myc. AutoDock analysis revealed that the binding effect of molecular leisure was evaluated by binding energy, and a binding energy <-5 kcal/mol was considered good. SMI decreased the mRNA and protein expression of c-Myc and HOXA5. SMI significantly inhibited the proliferative activity of KG1a and HL60 cells and induced their apoptosis. However, SMI had no significant effect on the cell cycle distribution of KG1a and HL60 cells. With increasing SMI concentrations, the p-p38/p38 ratio increased, while the protein expression of c-Myc and HOXA5 decreased, and the protein expression of cCaspase and cPARP increased. However, SB203580 intervention in addition to SMI reversed these changes. Tanshinone IIA, cryptanshinone and salvianolic acid B can bind to multiple sites of c-Myc. In summary, SMI could be used for the treatment of acute leukemia, and its mechanism may be related to activation of the p38MAPK signaling pathway.

Cytotoxicity of Doxorubicin-Curcumin Nanoparticles Conjugated with Two Different Peptides (CKR and EVQ) against FLT3 Protein in Leukemic Stem Cells.

A targeted micellar formation of doxorubicin (Dox) and curcumin (Cur) was evaluated to enhance the efficacy and reduce the toxicity of these drugs in KG1a leukemic stem cells (LSCs) compared to EoL-1 leukemic cells. Dox-Cur-micelle (DCM) was developed to improve the cell uptake of both compounds in LSCs. Cur-micelle (CM) was produced to compare with DCM. DCM and CM were conjugated with two FLT3 (FMS-like tyrosine kinase)-specific peptides (CKR; C and EVQ; E) to increase drug delivery to KG1a via the FLT3 receptor (AML marker). They were formulated using a film-hydration technique together with a pH-induced self-assembly method. The optimal drug-to-polymer weight ratios for the DCM and CM formulations were 1:40. The weight ratio of Dox and Cur in DCM was 1:9. DCM and CM exhibited a particle size of 20-25 nm with neutral charge and a high %EE. Each micelle exhibited colloidal stability and prolonged drug release. Poloxamer 407 (P407) was modified with terminal azides and conjugated to FLT3-targeting peptides with terminal alkynes. DCM and CM coupled with peptides C, E, and C + E exhibited a higher particle size. Moreover, DCM-C + E and CM-C + E showed the highest toxicity in KG-1a and EoL-1 cells. Using two peptides likely improves the probability of micelles binding to the FLT3 receptor and induces cytotoxicity in leukemic stem cells.

A biochemical assessment of apoptosis-inducing impact of Salinomycin in combination with ciprofloxacin on human leukemia KG1-a stem-like cells in the presence and absence of insulin.

Acute Myeloid Leukemia (AML) is a fast-developing invading cancer that impacts the blood and bone marrow, marked by the rapid proliferation of abnormal white blood cells. Chemotherapeutic agents, a primary treatment for AML, encounter clinical limitations such as poor solubility and low bioavailability. Previous studies have highlighted antibiotics as effective in inducing cancer cell death and potentially preventing metastasis. Besides, insulin is known to activate the PI3K/Akt pathway, often disrupted in cancers, leading to enhanced cell survival and resistance to apoptosis. In light of the above-mentioned points, we examined the anti-cancer impact of antibiotics Ciprofloxacin (CP) and Salinomycin (SAL) and their combination on KG1-a cells in the presence and absence of insulin. This was accomplished by exposing KG1-a cells to different doses of CP and SAL alone, in combination, and with or without insulin for 24-72h. Cell viability was evaluated using the MTT assay. Besides, apoptotic effects were examined using Hoechst staining and Annexin-V/PI flow cytometry. The expression levels of Bax, p53, BIRC5, Akt, PTEN, and FOXO1 were analyzed through Real-Time PCR. CP and SAL demonstrated cytotoxic and notable pro-apoptotic impact on KG1-a cells by upregulating Bax and p53 and downregulating BIRC5, leading to G0/G1 cell cycle arrest and prevention of the PI3K-Akt signaling pathway. Our findings demonstrated that combination of CP and SAL promote apoptosis in the KG1-a cell line by down-regulating BIRC5 and Akt, as well as up-regulating Bax, p53, PTEN, and FOXO1. Additionally, the findings strongly indicated that insulin effectively mitigates apoptosis by enhancing Akt expression and reducing FOXO1 and PTEN gene expression in the cells treated with CP and SAL. Our findings showed that the combined treatment of CP and SAL exhibit a strong anti-cancer effect on leukemia KG1-a cells. Moreover, it was discovered that the PI3K-Akt signaling can be a promising target in leukemia treatment particularly in hyperinsulinemia condition.

Hesperidin/Salinomycin Combination; a Natural Product for Deactivation of the PI3K/Akt Signaling Pathway and Anti-Apoptotic Factors in KG1a Cells.

AML is a highly aggressive malignant clonal disease of hematopoietic origin. Hesperidin as a polyphenol glycoside, Activates the apoptotic pathway and salinomycin as a k + selective ionophore. We examined how hesperidin and salinomycin induce pro-apoptotic effects in KG1a cells. Cells were divided into four groups; 1) control cells (CRTL), 2) cells treated with hesperidin 85μM, 3) cells treated with 2μM salinomycin, 4) cells treated with combination of salinomycin and hesperidin. The MTT assay was implemented to determine the IC50 of hesperidin and salinomycin in KG1a cell lines. Propidium iodide staining and flow cytometry were used to analyze the distribution of the cell cycle. The level of ROS was evaluated by fluorescent microscopy and spectrophotometry. Additionally, Akt, XIAP, Bad, and FOXO1 gene expression was analyzed by real-time PCR. Hesperidin/Salinomycin decreased the viability of KG1a leukemic cells more than Hesperidin and Salinomycin separately. Changes in the shape of apoptotic cells and rise in ROS levels were detected after Hesperidin/Salinomycin treatment. Our findings showed that following Hesperidin/Salinomycin treatment, the expression of PI3K/AKT signaling pathway related genes (AKT, PTEN and FOXO1), were in line with the destruction of KG-1a cells. Furthermore, XIAP and BAD mRNA were regulated to trigger apoptosis in cancer cells. The study discovered that hesperidin and salinomycin, could effectively hinder the PI3K/Akt signaling pathway in leukemia cancer cells. Also, the combination of hesperidin and salinomycin has the potential to be a treatment option for acute myeloid leukemia.

Cytokines secreted from bone marrow-derived mesenchymal stem cells promote apoptosis of CD34+ leukemic stem cells as anti-cancer therapy

ObjectiveThe effect of mesenchymal stem cells (MSCs) on the immortal characteristics of malignant cells, particularly hematologic cancer cells, remains a topic of debate, with the underlying mechanisms still requiring further elucidation. We explored the in vitro effect of the bone marrow-derived MSCs (BM-MSCs) on CD34+ leukemic stem cells (LSCs) enriched from the KG1-a cell line by assessing apoptosis, measuring cytokine levels, and examining TERT protein expression. Additionally, the potential signaling pathways implicated in this process, such as P53, PTEN, NF-κB, ERK1/2, Raf-1, and H-RAS, were also investigated. MethodsCD34+ LSCs were enriched from the KG1-a cell line with the magnetic activated cell sorting (MACS) method. Two cell populations (BM-MSCs and CD34+ LSCs) were co-cultured on trans well plates for seven days. Next, CD34+ LSCs were collected and subjected to Annexin V/PI assay, cytokine measurement, and western blotting. ResultsBM-MSCs caused a significant increase in early and late apoptosis in the CD34+LSCs. The significant presence of interleukin (IL)-2 and IL-4 was evident in the co-cultured media. In addition, BM-MSCs significantly increased the protein expression of P53, PTEN, NF-κB, and significantly decreased p-ERK1/2, Raf-1, H-RAS, and TERT. ConclusionThe mentioned effects of IL-2 and IL-4 cytokines released from BM-MSCs on CD34+ LSCs as therapeutic agents were applied by the components of P53, PTEN, NF-κB, p-ERK1/2, Raf-1, and H-RAS signaling pathways.

MicroRNA‑223 overexpression suppresses protein kinase C ε expression in human leukemia stem cell‑like KG‑1a cells.

MicroRNA-223 (miR-223) is dysregulated in various cancer types, including acute myeloid leukemia (AML). Despite this, there has been a lack of studies exploring the role of miR-223 in leukemic stem cells, particularly those involved in drug resistance, a major cause of chemotherapy failure in AML. The present study aimed to elucidate the impact of miR-223 on drug resistance in the leukemic stem-cell line, KG-1a. Two AML cell lines, KG-1 and KG-1a, differing in the proportion of CD34+CD38- cells, were assessed for doxorubicin (DOX) sensitivity using the Cell Counting Kit-8 assay. The expression levels of miR-223 and protein kinase C ε (PKCε) were evaluated via reverse transcription-quantitative PCR and western blot analysis. The association between miR-223 and its target, PKCε, was confirmed by luciferase activity assay. The effects of miR-223 overexpression and PKCε inhibition were also evaluated in KG-1a cells using miR-223 mimic and small interfering (si)RNA transfection, respectively. Daunorubicin was then used to assess drug sensitivity in the siRNA-transfected KG-1a cells. Compared with KG-1 cells, KG-1a cells displayed greater resistance to DOX, and had increased PKCε levels and decreased miR-223 expression. Overexpression of miR-223 led to PKCε protein downregulation in KG-1a cells, which was further confirmed by a luciferase assay demonstrating miR-223 targeting of PKCε. However, despite these effects, miR-223 overexpression and PKCε inhibition did not change drug sensitivity in KG-1a cells compared with negative control cells. In summary, the present study demonstrated that miR-223 could target and silence PKCε expression in KG-1a cells; however, the chemoresistance of KG-1a cells to anthracycline drugs may not be directly associated with the low expression of miR-223.

Metformin as an Enhancer for the Treatment of Chemoresistant CD34+ Acute Myeloid Leukemia Cells.

Acute myeloid leukemia is the second most frequent type of leukemia in adults. Due to a high risk of development of chemoresistance to first-line chemotherapy, the survival rate of patients in a 5-year period is below 30%. One of the reasons is that the AML population is heterogeneous, with cell populations partly composed of very primitive CD34+CD38- hematopoietic stem/progenitor cells, which are often resistant to chemotherapy. First-line treatment with cytarabine and idarubicin fails to inhibit the proliferation of CD34+CD38- cells. In this study, we investigated Metformin's effect with or without first-line conventional chemotherapy, or with other drugs like venetoclax and S63845, on primitive and undifferentiated CD34+ AML cells in order to explore the potential of Metformin or S63845 to serve as adjuvant therapy for AML. We found that first-line conventional chemotherapy treatment inhibited the growth of cells and arrested the cells in the S phase of the cell cycle; however, metformin affected the accumulation of cells in the G2/M phase. We observed that CD34+ KG1a cells respond better to lower doses of cytarabine or idarubicin in combination with metformin. Also, we determined that treatment with cytarabine, venetoclax, and S63845 downregulated the strong tendency of CD34+ KG1a cells to form cell aggregates in culture due to the downregulation of leukemic stem cell markers like CD34 and CD44, as well as adhesion markers. Also, we found that idarubicin slightly upregulated myeloid differentiation markers, CD11b and CD14. Treatment with cytarabine, idarubicin, venetoclax, metformin, and S63845 upregulated some cell surface markers like HLA-DR expression, and metformin upregulated CD9, CD31, and CD105 cell surface marker expression. In conclusion, we believe that metformin has the potential to be used as an adjuvant in the treatment of resistant-to-first-line-chemotherapy AML cells. Also, we believe that the results of our study will stimulate further research and the potential use of changes in the expression of cell surface markers in the development of new therapeutic strategies.

Emetine induces oxidative stress, cell differentiation and NF-κB inhibition, suppressing AML stem/progenitor cells

Acute myeloid leukemia (AML) is a fatal malignancy of the blood and bone marrow. Leukemic stem cells (LSCs) are a rare subset of leukemic cells that promote the development and progression of AML, and eradication of LSCs is critical for effective control of this disease. Emetine is an FDA-approved antiparasitic drug with antitumor properties; however, little is known about its potential against LSCs. Herein, we explored the antileukemic potential of emetine, focusing on its effects on AML stem/progenitor cells. Emetine exhibited potent cytotoxic activity both in hematologic and solid cancer cells and induced AML cell differentiation. Emetine also inhibited AML stem/progenitor cells, as evidenced by decreased expression of CD34, CD97, CD99, and CD123 in KG-1a cells, indicating anti-AML stem/progenitor cell activities. The administration of emetine at a dosage of 10 mg/kg for two weeks showed no significant toxicity and significantly reduced xenograft leukemic growth in vivo. NF-κB activation was reduced in emetine-treated KG-1a cells, as shown by reduced phospho-NF-κB p65 (S529) and nuclear NF-κB p65. DNA fragmentation, YO-PRO-1 staining, mitochondrial depolarization and increased levels of active caspase-3 and cleaved PARP (Asp214) were detected in emetine-treated KG-1a cells. Moreover, treatment with the pancaspase inhibitor Z-VAD(OMe)-FMK partially prevented the apoptotic cell death induced by emetine. Emetine treatment also increased cellular and mitochondrial reactive oxygen species, and emetine-induced apoptosis in KG-1a cells was partially prevented by the antioxidant N-acetylcysteine, indicating that emetine induces apoptosis, at least in part, by inducing oxidative stress. Overall, these studies indicate that emetine is a novel potential anti-AML agent with promising activity against stem/progenitor cells, encouraging the development of further studies aimed at its clinical application.

Bortezomib suppresses acute myelogenous leukaemia stem-like KG-1a cells via NF-κB inhibition and the induction of oxidative stress.

Acute myelogenous leukaemia (AML) originates and is maintained by leukaemic stem cells (LSCs) that are inherently resistant to antiproliferative therapies, indicating that a critical strategy for overcoming chemoresistance in AML therapy is to eradicate LSCs. In this work, we investigated the anti-AML activity of bortezomib (BTZ), emphasizing its anti-LSC potential, using KG-1a cells, an AML cell line with stem-like properties. BTZ presented potent cytotoxicity to both solid and haematological malignancy cells and reduced the stem-like features of KG-1a cells, as observed by the reduction in CD34- and CD123-positive cells. A reduction in NF-κB p65 nuclear staining was observed in BTZ-treated KG-1a cells, in addition to upregulation of the NF-κB inhibitor gene NFΚBIB. BTZ-induced DNA fragmentation, nuclear condensation, cell shrinkage and loss of transmembrane mitochondrial potential along with an increase in active caspase-3 and cleaved PARP-(Asp 214) level in KG-1a cells. Furthermore, BTZ-induced cell death was partially prevented by pretreatment with the pancaspase inhibitor Z-VAD-(OMe)-FMK, indicating that BTZ induces caspase-mediated apoptosis. BTZ also increased mitochondrial superoxide levels in KG-1a cells, and BTZ-induced apoptosis was partially prevented by pretreatment with the antioxidant N-acetylcysteine, indicating that BTZ induces oxidative stress-mediated apoptosis in KG-1a cells. At a dosage of 0.1 mg/kg every other day for 2 weeks, BTZ significantly reduced the percentage of hCD45-positive cells in the bone marrow and peripheral blood of NSG mice engrafted with KG-1a cells with tolerable toxicity. Taken together, these data indicate that the anti-LSC potential of BTZ appears to be an important strategy for AML treatment.

Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.

Abstract 5227: Gamma-delta (γδ) CAR-T cells lacking the CD3ζ signaling domain enhance targeted killing of AML cells and preserve healthy tissues

Abstract Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy against B cell malignancies, offering hope to patients with limited treatment options. However, extending this therapy to myeloid malignancies and solid tumors has proven challenging due to co-expression of targetable antigens on hematopoietic progenitors and healthy tissues. In this context, γδ T cells show promise, as they can directly identify and eliminate malignant cells via recognition of multiple tumor-associated stress antigens rarely expressed on normal tissues. We leveraged the tumor-sensing capabilities of γδ T cells with enhanced tumor localization by employing a non-signaling CAR (nsCAR) that excludes the CD3ζ domain, facilitating targeted tumor cell killing while sparing healthy tissues. We designed second-generation lentiviral constructs for anti-CD33-scfv for in vitro evaluation against acute myeloid leukemia (AML) lines HL-60, KG-1a, and MOLM-13 and the chronic myeloid leukemia (CML) line K-562 Additionally, we tested CD33/CD123 dual-targeting nsCAR constructs (ns-dCAR) to determine if the addition of CD123 targeting enhanced the therapeutic index. We also tested whether a secreted IL-15 (sIL-15) would enhance CAR-T cell fitness. We observed significant upregulation of CD69 in Jurkat cells transduced with signaling CARs in co-culture with KG-1a cells but not in those with nsCAR constructs. We also noticed a time-dependent reduction (43%) in the CD3ζ+ CD33CAR+ population over a 7-day extended coculture while the nsCAR+ Jurkat population remained stable, suggesting potential mitigation of activation-induced cell death (AICD). Expanded and activated γδ T cells from healthy donors were then transduced with nsCAR lentiviral vectors. The ns33CAR+ γδ T cells exhibited enhanced killing capability against HL-60 (up to 1.3x) and K-562 (up to 1.6x) compared to unmodified γδ T cells (UTD) in a 24-hour cytotoxicity assay. Incorporation of sIL-15 into the nsCD33 CAR construct also increased killing across all 4 cell lines (up to 1.8x for HL-60, up to 2.6x for KG-1a, up to 2.0x for MOLM-13 and up to 2.0x for K-562). Minimal cytotoxicity (&amp;lt;10%) was observed for nsCARs or UTDs against normal CD33+/CD123+ cells from healthy donor PBMC or CD34+ HPSCs. The ns-dCAR constructs did not improve in vitro AML killing compared to ns33CAR alone, although ns-dCARs generally exhibited lower transduction efficiency. In summary, our findings suggest that combining nsCAR constructs with γδ T cells may widen the therapeutic window to expand the reach of CAR-T therapy to cancers with limited target antigen expression on critical healthy tissues. Further optimization to improve integration and incorporation of membrane-bound IL-15 co-expression holds the potential to enhance both the efficacy and safety profiles of next-generation adoptive cell therapies against a broader spectrum of cancers. Citation Format: Lei Ding, Yanjie Li, Jiexue Li, Mariska Ter Haak, Lawrence Lamb. Gamma-delta (γδ) CAR-T cells lacking the CD3ζ signaling domain enhance targeted killing of AML cells and preserve healthy tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5227.

Enhanced Expression of Glycolytic Enzymes and Succinate Dehydrogenase Complex Flavoprotein Subunit A by Mesothelin Promotes Glycolysis and Mitochondrial Respiration in Myeloblasts of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by rapid growth and uncontrolled proliferation of undifferentiated myeloid cells. Metabolic reprogramming is commonly observed in the bone marrow of AML patients, as leukemia cells require increased ATP supply to support disease progression. In this study, we examined the potential role of mesothelin as a metabolic modulator in myeloid cells in AML. Mesothelin is a well-known marker of solid tumors that promotes cancer cell proliferation and survival. We initially analyzed alterations in mesothelin expression in the myeloblast subpopulations, defined as SSC-Alow/CD45dim, obtained from the bone marrow of AML patients using flow cytometry. Our results showed overexpression of mesothelin in 34.8% of AML patients. Subsequently, metabolic changes in leukemia cells were evaluated by comparing the oxygen consumption rates (OCR) of bone marrow samples derived from adult AML patients. Notably, a higher OCR was observed in the mesothelin-positive compared to the mesothelin-low and non-expressing groups. Treatment with recombinant human mesothelin protein enhanced OCR and increased the mRNA expression of glycolytic enzymes and mitochondrial complex II in KG1α AML cells. Notably, siRNA targeting mesothelin in KG1α cells led to the reduction of glycolysis-related gene expression but had no effect on the mitochondrial complex gene. The collective results demonstrate that mesothelin induces metabolic changes in leukemia cells, facilitating the acquisition of a rapid supply of ATP for proliferation in AML. Therefore, the targeting of mesothelin presents a potentially promising approach to mitigating the progression of AML through the inhibition of glycolysis and mitochondrial respiration in myeloid cells.

Inhibitory Effect of Metformin and Arsenic Trioxide on KG1a Cell Proliferation

To investigate the effect of metformin and arsenic trioxide on KG1a cells proliferation of acute myeloid leukemia and its possible mechanism. CCK-8 method was used to detect the killing effect of metformin, arsenic trioxide and combined application on KG1a cells. Annexin V-FITC/PI Dual Stain Flow Cytometry was used to detect the effect of combined application on apoptosis of KG1a cells. Western blot was used to detect the expression of intracellular apoptosis-,autophagy-related protein. Metformin and arsenic trioxide alone or in combination could inhibit the proliferation of KG1a cells and induce apoptosis of KG1a cells, and the proliferation inhibition rate and apoptosis rate in the combined drug group were higher than those in the drug group alone(P <0.05). The combination of drugs induced upregulation of Caspase 8 protein and P62 protein expression and was higher than that in the drug group alone(P <0.05). Metformin can synergize with arsenic trioxide to kill KG1a cells, and its mechanism of action may be related to inducing apoptosis and enhancing autophagy.

Exploring the Potential of siRNA Delivery in Acute Myeloid Leukemia for Therapeutic Silencing.

We investigated the feasibility of using siRNA therapy for acute myeloid leukemia (AML) by developing macromolecular carriers that facilitated intracellular delivery of siRNA. The carriers were derived from low-molecular-weight (<2 kDa) polyethyleneimine (PEI) and modified with a range of aliphatic lipids. We identified linoleic acid and lauric acid-modified PEI as optimal carriers for siRNA delivery to AML cell lines KG1 and KG1a, as well as AML patient-derived mononuclear cells. As they have been proven to be potent targets in the treatment of AML, we examined the silencing of BCL2L12 and survivin and showed how it leads to the decrease in proliferation of KG1 and stem-cell-like KG1a cells. By optimizing the transfection schedule, we were able to enhance the effect of the siRNAs on proliferation over a period of 10 days. We additionally showed that with proper modifications of PEI, other genes, including MAP2K3, CDC20, and SOD-1, could be targeted to decrease the proliferation of AML cells. Our studies demonstrated the versatility of siRNA delivery with modified PEI to elicit an effect in leukemic cells that are difficult to transfect, offering an alternative to conventional drugs for more precise and targeted treatment options.

Cryptanshinone extract of Salvia miltiorrhiza stimulates pediatric acute myeloid leukemia stem cell apoptosis and the anti-inflammatory mechanism via accelerating microRNA-211-5p to supress Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway activation.

This study was designed to explore cryptanshinone (CPT) extract of Salvia miltiorrhiza stimulating pediatric acute myeloid leukemia (AML) stem cell (LSC) apoptosis and anti-inflammatory mechanism via accelerating microRNA (miR)-211-5p to restrain Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway activation. Obtaining blood samples from pediatric acute myeloid leukemia patients and healthy volunteers and detecting miR-211-5p and JAK2 were performed. Purchase of the human AML cell line KG1a was conducted, and sorting of KG1a cells was to gain LSC. Test of miR-211-5p and JAK2, the phosphorylation of JAK2/STAT3 was implemented. Pretreatment of LSCs was with CPT. Variation of miR-211-5p and JAK2 in LSCs was via plasmid transfection to explore their actions in cell advancement with apoptosis and inflammation. Identification of the targeting of miR-211-5p with JAK2 was implemented. In results: MiR-211-5p was declined in endometrial cancer, while JAK2 was elevated; CPT was available to boost LSC apoptosis and restrain the inflammation; elevated miR-211-5p or repressive JAK2 was available to strengthen the acceleration of CPT on LSCs apoptosis and the repression of inflammation; MiR-211-5p targeted JAK2; augmented JAK2 was available to turn around the action of elevated miR-211-5p. We conclude that CPT extract of Salvia miltiorrhiza stimulated pediatric LSC apoptosis and restrained the inflammation via accelerating microRNA (miR)-211-5p to suppress JAK2/STAT3 pathway activation.

A Novel Anticancer Quinolone, ( R)-Wac-224 Monotherapy or Combination with Cytarabine/Venetoclax Confers Promising Activities Against Acute Myeloid Leukemia

Introduction Approximately 60%-80% of patients with acute myeloid leukemia (AML) who achieve complete remission eventually relapse after conventional chemotherapy and have poor prognoses despite the recent advances of novel anticancer agents. Targeting topoisomerase 2 (Top2) is important to treat patients with AML, as anthracycline is widely used in general practice. However, anthracycline-induced cardiotoxicity or anthracycline resistance due to P-glycoproteinoverexpression often becomes major obstacles to successful treatment. Therefore, continuing development of more effective novel treatments for AML is necessary. Quinolones, antibacterials that attack DNA gyrase and topoisomerase IV (functional analog of mammalian Top2), possess a broad spectrum chemotherapeutic activities including anti-leukemic effects (e.g. vosaroxin). In this study, we developed and characterized a novel quinolone that demonstrates promising activities against human AML as a single agent and in combination with other agents. Methods and Results We developed 5-methyl-7-{3-[(oxan-2-ylmethyl) carbamoyl] azetidin-1-yl}-4-oxo-1-(1,3-thiazol-2-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid [racemic WAC-224 (WAC) or (R)-WAC-224 (R-WAC), which is a novel anticancer quinolone, targeting Top 2. WAC inhibited Top2α comparable with vosaroxin (a known Top2 inhibitor) in a topoisomerase relaxation assay. IC 50 of vosaroxin and WAC were 1.10 μM and 2.26 μM, respectively. R-WAC significantly inhibited the human AML cell line proliferation (MV4-11, HL60, and KG1a), which was comparable to daunorubicin and cytarabine, in a manner not affected by P-glycoprotein overexpression. WAC also elicited cell apoptosis against MV4-11 cells comparable with vosaroxin. Whereas, WAC did neither increase serum troponin-T nor decrease the crypt numbers in the small intestine, indicating WAC was less toxic than doxorubicin. R-WAC monotherapy demonstrated prolonged survival in the AML mice model and inhibited tumor growth in the MV4-11 xenograft mice model. Moreover, the combination of R-WAC and cytarabine demonstrated more active anti-leukemia effects than daunorubicin and cytarabine in the AML mice model. R-WAC and cytarabine or daunorubicin and cytarabine did not exhibited decreased body weight in mice. Next, we investigated R-WAC combination with venetoclax, selective BCL-2 inhibitor, for anti-leukemia effects. R-WAC and venetoclax exhibited increased cell apoptosis against MV4-11 and KG1a cells in vitro, and more active anti-leukemia effects in xenotranplant MV4-11 mouse model than each monotherapy. These results suggest that R-WAC and combination with cytarabine or venetoclax has strong anti-leukemia effects without increased toxicities. Finally, R-WAC significantly suppressed the colony-forming capacity of bone marrow CD34 + stem/progenitor cells in refractory AML patients, indicating R-WAC depleted AML stem cells. Conclusion R-WAC has promising anti-leukemia effects with less toxicities compared with conventional anthracycline possibly due to the different inhibitory mechanisms of Top2 isoform (anthracycline inhibits Top2β). These results indicate that R-WAC is a promising therapeutic agent for AML and support its clinical development, especially as a combination of R-WAC with cytarabine or venetoclax.

CX43-Mediated Mitochondria Transfer Enhances the Stemness of Leukemia Stem Cells Though Metabolic Remodeling

Background :Despite improvements in treatment options for acute myeloid leukemia (AML), chemotherapy resistance remains an important clinical concern with a high recurrence rate in patients.Leukemia stem cells (LSCs) play a decisive role in the relapse resistance of leukemia. Bone marrow mesenchymal stem cells(BMSCs) protect LSCs from being completely removed from the bone marrow, but the underlying mechanisms are poorly understood.In this study, we aimed to uncover the mechanism of BMSCs-regulated the stemness of leukemia stem cells through mitochondrial transfer mediated by Connexin43(CX43), thus providing a promising clinical therapy target. Methods: Primary BMSCs were isolated from the primary bone marrow specimens of patients with AML and cultured in MSCM culture base within 10% serum.CX43 overexpressing BMSCs (CX43-BMSCS) were constructed by lentivirus transfection.The efficiency of CX43 overexpression was verified by immunofluorescence, qPCR and Western Blot.In vitro, CX43-BMSCs and control BMSCs (EV-BMSCs) were cocultured with KG-1a cells.In some experiments,vincristine (1nM) was added to inhibit the mitochondrial transferring.Bidirectional mitochondrial transfer was detected by flow cytometry and confocal microscopy.The mechanism of mitochondria transfer was analyzed by inhibitor assays.The proliferation of KG-1a from the coculture system were assessed by Flow cytometry and EdU assay.After co-culture, the clone formation of KG-1a was detected by using semisolid cloning assay.The mitochondrial respiration of KG-1a was analyzed by using Seahorse.The transcriptomes of KG-1a after co-culture using RNA-seq.Finally,we verified our findings using LC-MS non-targeted metabolomics to detect the metabolites changes of KG-1a after co-culture. Results: Compared with Control-BMSCs and EV-BMSCs,the mRNA and protein expression levels of CX43 in CX43-MSCs were significantly increased (P&amp;lt;0.05).In the co-culture system without serum for 48h,BMSCs transfer mitochondria to KG-1a cells but receive no mitochondria from KG-1a.Moreover,CX43-BMSCs transferred more mitochondria to KG-1a than EV-BMSCs group (P&amp;lt;0.05),while there was no obvious mitochondrial transfer was observed in the transwell and 10% serum co-culture system.In addition,compared with EV-BMSCs,CX43-BMSCs significantly increased the proliferation and adhesion of KG-1a (P&amp;lt;0.05) and clone formation ability (P&amp;lt;0.05).Vincristine significantly reduced the mitochondrial transfer from CX43-BMSCs to KG-1a,and decreased the adhesion of CX43-BMSCs to KG-1a,as well as the proliferation and clone formation of KG-1a after co-culture.The metabolic capacity of KG-1a cells in CX43-BMSCs was significantly increased compared with EV-BMSCs (P&amp;lt;0.05),and this metabolic enhancement could be reversed by vincristine.Hierarchical cluster analysis (HC) of metabolites showed that there were significant differences in metabolites among treatment groups (p&amp;lt;0.05),showed unique metabolic characteristics related to CX43-induced changes.The GO analysis revealed that the enrichments occurring in KG-1a after co-cultured with CX43-BMSCs mainly included cell adhesion and migration,cell proliferation and regulation,signal transduction,PI3K/Akt pathway,transmembrane transport and intracellular protein metabolism.Metabolism-related pathways,like nucleotide metabolism,lipid metabolism and amino acid metabolism,were enriched in KG-1a after co-cultured with CX43-BMSCs.Furthermore,KG-1a after co-cultured with CX43-BMSCs showed up-regulated of promoting the proliferation and the stemness of AML-LSCs,including N-cadherin (CDH2),CD244,SLC43A2,CYBB and IGLL1. Conclusion: We show evidence that CX43 mediated mitochondria transfer from BMSCs to LSCs which promoting the adhesion of BMSCs to LSCs under nutrient deficiency condition,and then enhanced the proliferation and cloning ability of LSCs,altered gene expression profiles related to cell proliferation,adhesion,and energy metabolism,and increased the expression of genes related to maintaining stemness of LSCs, allows metabolic remodeling to regulate LSCs stemness, may be a potential therapeutic target for AML treatment. Keywords:Leukemia stem cells;Bone marrow microenvironment;Connexin;Mitochondrial transfer;Metabolic remodeling

Combining Allogeneic Gamma Delta T Cells with a Phosphoantigen Prodrug Promotes Apoptosis of Acute Myeloid Leukemia Stem and Progenitor Cells

Despite recent therapeutic advances, patients with relapsed/refractory acute myeloid leukemia (r/r AML) have limited treatment options and poor prognosis. Leukemic stem cells (LSCs) are considered as relapse-initiating cells and the “culprit” of treatment resistance. Allogeneic and autologous gamma delta (γδ) T cell-based therapies have been explored for a long time and proven to be safe in patients through multiple clinical trials. However, quite a few AML blasts obtained from r/r AML patients show resistance to γδ T cell-mediated cytotoxicity. Novel strategies targeted LSCs may improve efficacy of γδ T cell-mediated r/r AML therapy. Given that γδ T cells exert antitumor capacity via multiple pathways, to identify the way matters most in the resistance of γδ T cell-mediated cytotoxicity, we examined the median fluorescence intensity (MFI) of the ligands expressed in tumor cells by flow cytometry, including butyrophilin 3A (BTN3A), butyrophilin 2A1 (BTN2A1), the human MHC class I chain-related genes (MICA and MICB), the unique-long 16 binding proteins (ULBP1, ULBP2/5/6, ULBP3), Fas, the receptor of TNF-related apoptosis-inducing ligand (TRAIL) DR5, CD112 and CD155. The cytotoxicity activity of γδ T cells was measured by flow cytometry-based killing assay or bright-lite luciferase assay system after coculturing with AML cell lines and blasts at an appropriate effector to target (E:T) ratio. Then we employed a Support Vector Machine model to predict the killing efficiency and identified that BTN3A and BTN2A1 play the predominant role in γδ T cell-mediated cytotoxicity based on the Shapley additive explanation. BTN3A and BTN2A1 are the key ligands in the γδ TCR-phosphoantigen (pAg) recognition activity. PAgs, which are produced in tumor cells function as “molecular glues” to promote heteromeric association of BTN3A1 and BTN2A1 (Yuan L et al., BioRxiv 2022). To promote the γδ T cell-mediated cytotoxicity targeting r/r AML, here we synthesized a pAg prodrug, which show great plasma stability and membrane permeability. We anticipate this small molecule would lead pAg accumulation in tumor cells and cause increased γδ T cell activation. To identify the killing efficacy of pAg prodrug-induced γδ T cell activation, we select KG-1α (a human leukemia stem cell-like cell) as target cell. Zoledronate (ZOL), which is employed to stimulate γδ T cells in multiple γδ T cell-based clinical trials, is used in our killing assay as compared group. As expected, combining γδ T cells with pAg prodrug or ZOL both significantly improved the percent of specific killing when the E:T ratio is 1:1. The pAg prodrug showed more potent activity than ZOL group with EC50 value of 0.1 nM vs 17.93μM. And the specific killing percent of pAg prodrug group is 83.26±2.1 % (1 nM), comparing with negative control 15.17±3.4 % (only γδT cells). Futhermore, at relatively low E:T ratio (1:4), γδ T cells with pAg prodrug remain excellent cytotoxicity when targeting MV411 cell and OCI-AML3 cell, which are widely used in r/r AML research. After coculturing with primary leukemia stem and progenitor cells (CD34 +) obtained from r/r AML patients for 6~8 h, the apoptosis percentage of CD34 + leukemia cells was 42.34% comparing with negative control (only γδT cells) 2.53%. Then we evaluated the in vivo efficacy of combining γδT cells and pAg prodrug in NSG mice bearing MV411 cells. Compared with only γδT cells-treated group, combination with pAg prodrug presented better tumor clearing. To elucidate the pathway of pAg prodrug-mediated killing, we tested mitochondria apoptosis related events. After treated by γδ T cells with pAg prodrug, KG-1α cells showed loss of mitochondrial outer membrane potential and increasingly actived caspase 3/7. Meanwhile, pAg prodrug directly activates effector γδ T cells with increased secretion of CD107a and IFN-γ. Considering the possibility of toxicity towards normal hematopoietic stem and progenitor cells, we conducted colony-forming units (CFUs) assay. After treatment with γδT cells and pAg prodrug, the number of CFUs of CD34 + cells from cord blood was not effected compared with the CD34 + cells only group. Overall, we explored the predominant factor in γδ T cell-mediated killing and synthesized a pAg prodrug to improve the killing efficacy. We demonstrated that combining γδ T cells with pAg prodrug has effectively promoted apoptosis of AML stem and progenitor cells sparing normal hematopoietic stem and progenitor cells.

Synergy of Ruxolitinib and Carfilzomib in Targeting the PAX5::JAK2 fusion I n Vitro: Potential Therapeutic Advantage for a Subset of Ph-like Acute Lymphoblastic Leukemia

INTRODUCTION PAX5::JAK2 is a gene fusion that drives a subset of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) and confers aggressive disease, poor prognosis and poor response to treatment. PAX5::JAK2 is present in approximately 12% of Ph-like ALL cases. Targeted therapy is urgently required to improve outcomes. We evaluated the efficacy of JAK inhibitor ruxolitinib (RUX) and proteosome inhibitors carfilzomib (CFZ) and bortezomib (BTZ) as monotherapy and in combination against PAX5::JAK2 transfected Ba/F3 cells in vitro. Downregulation of proteosome genes via reduction in STAT3 phosphorylation is a shared mechanism of RUX and proteosome inhibitors. Given that the PAX5::JAK2 fusion results in increased STAT3 phosphorylation driving proliferation, it was proposed that the combination of proteosome inhibitors and RUX would demonstrate synergy in inhibiting proliferation of PAX5::JAK2 driven leukemic cells. METHODS Mouse derived Ba/F3 cells, an IL-3 dependent B-ALL model, were transfected with the PAX5::JAK2 fusion and demonstrated IL-3 independence. We applied, as single treatments, differing concentrations of RUX and the proteosome inhibitors CFZ and BTZ to PAX5::JAK2 transfected Ba/F3 cells and two control cell lines - IL-3 dependent empty vector Ba/F3 cells and an IL-3 independent KG1a myeloid cell line. Differing concentrations of CFZ and 200nM of RUX were then applied in combination. 200nM of RUX was selected because it is a dose at which the drug has minimal effect as a single agent. Inhibitors were applied to cells for 72 hours then percentage of live and dead cells at each concentration was ascertained by flow cytometry using Annexin V and LIVE/DEAD fixable aqua dead cell stain. Downstream STAT5 and STAT3 activation in basal state, and as a response to treatment, was assessed using phosphoflow. We demonstrated mechanisms of growth inhibition via reduction in STAT5 and STAT3 phosphorylation by the inhibitors. RESULTS RUX, CFZ and BTZ were all effective against PAX5::JAK2 transfected Ba/F3 cells with a median lethal dose (LD50) of 514nM (CI 498-530nM) for RUX, 38nM (CI 36-39nM) for CFZ and 19nM (CI 18-20nM) for BTZ. RUX had a variable effect on IL-3 dependent empty vector control cells which also demonstrate JAK/STAT activation, but no effect on the IL-3 independent KG1a cells. CFZ had a lesser effect on empty vector control cells with a LD50 of 59nM (CI 58-60nM) and no effect on KG1a control cells. BTZ had a significant effect on both control cell lines, predicting off-target toxicity and consequently was not applied in combination with RUX. RUX and CFZ in combination demonstrated an LD50 of 21nM (CI 18-25nM) (see figure 1) which is significantly lower than the LD50s for either agent used as monotherapy. Combination index for RUX and CFZ at 45nM was 0.80 and at 50nM was 0.49, suggesting moderate synergy. Phosphoflow demonstrated reduced pSTAT5 with the application of RUX and CFZ to PAX5::JAK2 transfected Ba/F3 cells with mean fluorescence intensity (MFI) reducing from 351 to 285 in inhibitor treated cells, while for STAT3 MFI reduced from 396 to 249. (see figure 2) There was no effect on MFI for control cell lines. This demonstrates that the mechanism for growth inhibition is inhibition of JAK/STAT signaling due to the reduction in pSTAT3 and pSTAT5. CONCLUSIONS We showed in vitro that RUX, CFZ and BTZ are all potent against the high risk PAX5::JAK2 fusion. RUX and CFZ in combination demonstrated moderate synergy against PAX5::JAK2 and inhibited proliferation through their effect on JAK/STAT pathways. The RUX/CFZ combination, drugs widely available in clinical practice, is particularly promising given the apparent synergistic effect on limiting leukemic cell growth. Such treatments warrant clinical trials for management of the poor prognostic group of Ph-like ALL with PAX5::JAK2, given that they may enhance efficacy and lessen toxicity. Clinical trials have already demonstrated efficacy and safety of RUX and CFZ as single agents in combination with chemotherapy for the treatment of B-ALL which indicates the feasibility of combining the two agents clinically in Ph-like ALL.

Production of recombinant human long-acting IL-18 binding protein: inhibitory effect on ulcerative colitis in mice.

Interleukin-18 binding protein (IL-18BP) is a natural IL-18 inhibitor in vivo, which can effectively neutralize IL-18 and inhibit the inflammatory signaling pathway induced by IL-18, thus playing an anti-inflammatory role. Traditional production methods primarily rely on eukaryotic animal cell expression systems, which often entail complex processes, lower yields, and increase production costs. In this study, we present a novel approach for expressing IL-18BP fusion protein using the Escherichia coli (E. coli) system. The N-terminal segment of IL-18BP was fused with the small ubiquitin-related modifier (SUMO) tag, enabling soluble expression, while the C-terminal segment was fused with the human IgG1 Fc fragment to prolong its in vivo lifespan. Through screening, we obtained a high-expression engineering strain from a single colony and developed optimized protocols for fermentation and purification of the recombinant SUMO-IL-18BP-Fc protein. The SUMO tag was subsequently cleaved using SUMO protease, and the purified recombinant human IL-18BP-Fc (rhIL-18BP-Fc) exhibited a purity exceeding 90% with a yield of 1 g per liter of bacterial solution. The biological activities and underlying mechanisms of rhIL-18BP-Fc were evaluated using cell lines and a mouse model. Our results demonstrated that rhIL-18BP-Fc effectively inhibited IL-18-stimulated IFN-γ production in KG-1a cells in vitro and ameliorated DSS-induced ulcerative colitis in mice. In conclusion, we successfully employed the SUMO fusion system to achieve high-level production, soluble expression, and prolonged activity of rhIL-18BP-Fc in E. coli. These findings lay the groundwork for future large-scale industrial production and pharmaceutical development of rhIL-18BP-Fc protein. KEY POINTS: • Effective expression, fermentation, and purification of bioactive rhIL-18BP-Fc protein in E. coli. • The rhIL-18BP-Fc protein has a great potential for the therapy of ulcerative colitis by inhibiting the expression of inflammatory factors.