Cell Lines KG-1 Research Articles

Up-Regulation of miR-625-5p Correlates with Suppressed Sox2, Increased Apoptosis, and Cell Cycle Arrest via The PI3K/AKT Signalling Pathway in Acute Myeloid Leukaemia

Background: Up-regulation of the microRNA-625 and abnormal expression of the Sox2 gene have been studied and seen in several tumors. Few reports have also shown the aberrant expression of miR-625 and Sox2 expression in various cancers. Several studies have also confirmed that phosphatidylinositol 3' -kinase /protein kinase B pathways regulate hematological malignancies, including Acute Myeloid Leukemia (AML). Thus, this study aimed to investigate the effects of mir-625 up-regulation on proliferation, apoptosis, and cell cycle by targeting the Sox2 gene via the downstream Akt signaling pathway and cell cycle regulators, such as p21, p27, and cyclin E in the KG-1 cell line. Materials and Methods: Cells obtained from the KG-1 cell line were cultured and transfected with plasmid DNA (miR-625) and scrambled as the control using the Lonza electroporation system. Flow cytometry was used to evaluate cell cycle, proliferation, and apoptosis. Relative gene expression was validated by qRT-PCR. All data were analyzed using graph pad prism 7.01 and REST 2009. Results: KG-1 cells transfected with the mir625-GFP construct showed decreased proliferation, increased apoptosis, and induced cell cycle arrest. Low levels of Sox2, p21, cyclin E, and up-regulation of p27 were confirmed and validated by qRT-PCR ( P < 0.05 ). Conclusion: MiR-625 can be a promising approach to aid in the treatment of AML. However, further studies are required in this field

Atranorin is a novel potential candidate drug for treating myelodysplastic syndrome

This work is devoted to the study of biocompatibility, cyto- and genotoxicity, mechanism of action and prospects for the use of atranorin, which is an AKT kinase inhibitor, for the treatment of myelodysplastic syndrome. Atranorin was isolated by preparative flash chromatography; identification was carried out by UV, IR, and NMR spectroscopy, mass spectrometry, and elemental analysis. Biocompatibility studies included studies of haemocompatibility, genotoxicity, antioxidant activity, cytotoxicity against ECV340 and HEK293 cell lines. Computer modelling of the interaction of atranorin with AKT kinase was carried out using docking followed by molecular dynamics of the resulting complexes; the ADMET properties of atranorin were also calculated. Flow cytometry included analysis of the expression level of PD-L1 and TIM-3 in the presence of atranorin on THP-1, Mono-Mac-1 and KG-1 cell lines, as well as human bone marrow cells.

Antileukemia Activity of Human Natural Killer Cell-Derived Nanomagic Bullets against Acute Myeloid Leukemia (AML).

Background: Cancer is among the serious health problems of the medical world, for treatment of which severe treatments are used. However, the prognosis of cancer patients is still poor. The application of NK cell-derived exosomes (NK-Exo) is a new method for cancer immunotherapy. These nanoparticles with a size range of 30-120 nm are a small model of mother cells. In this study, the anti-tumor activity of NK-Exo and LAK-Exo (activated NK cell-derived exosome) against acute myeloid leukemia (AML) is investigated in vitro. Materials and Methods: The MACS method was performed for the separation of NK cells from the buffy coats of healthy donors, and an EXOCIBE kit was used for the isolation of NK-Exo. After treating the KG-1 cell line with different doses of NK-Exo, MTT assay, and annexin V-PE were done to evaluate cell proliferation and apoptosis, respectively, and for confirmation of involved proteins, Real-Time PCR and western blotting were performed. Results: Anti-tumor activity of NK-Exo and LAK-Exo was dose- and time-dependent. Their highest activities were observed following 48 hours of incubation with 50 µg/ml exosome (p<0.0001). However, this cytotoxic activity was also seen over a short period of time with low concentrations of NK-Exo (p<0.05) and LAK-Exo (p<0.001).The cytotoxic effect of LAK-Exo on target cells was significantly higher than NK-EXO. The induction of apoptosis by different pathways was time-point dependent. Total apoptosis was 34.56% and 51.6% after 48 hours of tumor cell coculture with 50µg/ml NK-Exo and LAK-Exo, respectively. Significant expression of CASPASE3, P38, and CYTOCHROME C genes was observed in the cells treated with 50 µg/ml NK-Exo and LAK-Exo. Conclusion: Our study confirmed the antileukemia activity of NK-Exo against AML tumor cells in vitro. Therefore, NK-Exo can be considered as a promising and effective treatment for leukemia therapy.

Venetoclax efficacy on acute myeloid leukemia is enhanced by the combination with butyrate

Venetoclax has been approved recently for treatment of Acute myeloid leukemia (AML). Venetoclax is a BH3-mimetic and induces apoptosis via Bcl-2 inhibition. However, venetoclax’s effect is still restrictive and a novel strategy is needed. In the present study, we demonstrate that sodium butyrate (NaB) facilitates the venetoclax’s efficacy of cell death in AML cells. As a single agent, NaB or venetoclax exerted just a weak effect on cell death induction for AML cell line KG-1. The combination with NaB and venetoclax drastically induced cell death. NaB upregulated pro-apoptotic factors, Bax and Bak, indicating the synergistic effect by the collaboration with Bcl-2 inhibition by venetoclax. The combined treatment with NaB and venetoclax strongly cleaved a caspase substrate poly (ADP-ribose) polymerase (PARP) and a potent pan-caspase inhibitor Q-VD-OPh almost completely blocked the cell death induced by the combination, meaning that the combination mainly induced apoptosis. The combination with NaB and venetoclax also strongly induced cell death in another AML cell line SKNO-1 but did not affect chronic myeloid leukemia (CML) cell line K562, indicating that the effect was specific for AML cells. Our results provide a novel strategy to strengthen the effect of venetoclax for AML treatment.

Comparing the Effects of Free and Liposomal Indole Compounds on Bax and Bcl2 Gene Expression Changes in the KG-1 Cell Line

Background: For patients with acute myeloid leukemia (AML), the long-term survival rate is still very low. This study examines the effects on AML cell lines of an indole chemical in its free and liposomal forms. Material and Method: In this experimental case control study, an AML-originated KG-1 cell line was cultured in RPMI 1640 medium. The cells were treated with the free and liposomal forms of an indole compound (C18H10N2F6O) at different concentrations of 20, 40, 100, 200, and 400 µg/mL after they attained the proper confluence. The cellular metabolic activity was examined by an MTT assay. The expression of BAX and BCL-2 genes was investigated by q-PCR to assess the apoptotic effect of that compound. The analysis was also done between each experimental group and the control group using t-test. P&lt;0.05 was assumed significant. Results: Based on the MTT assay, the lethal effective dose of free indole was found to be 245.1 µg/ml and 164.8 µg/ml in 24 and 48 hours, respectively. The corresponding values for liposomal indole were 47.2 µg/ml and 40.6 µg/ml. Furthermore, treatment with free and liposomal forms of indole resulted in a decline in the expression level of the BCL-2 gene. However, in the case of the liposomal compound, this decrease was only statistically significant after 48 hours of treatment (P &lt; 0.05). Furthermore, the expression of BAX gene increased after treatment with both free and liposomal forms of indole, but it significantly increased only after treatment with the liposomal compound (p &lt; 0.05). Conclusion: These results suggest that an indole derivative, especially when liposomal, causes apoptosis in AML cells, hence exhibiting cytotoxic effects. To confirm the potential usefulness of this indole derivative as a therapeutic agent for inhibiting tumor progression in the setting of human malignancies, more studies on physiologically relevant models are necessary.

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.

Effect of Long Non-Coding RNA LINC01268 on the Malignant Biological Behaviors of Acute Myeloid Leukemia Cells

To investigate the effect of long non-coding RNA LINC01268 on apoptosis of acute myeloid leukemia (AML) cells and related mechanisms. The expression levels of LINC01268 and miR-217 in peripheral blood samples from AML patients and AML cell lines HL-60 and KG-1 were detected by qRT-PCR. HL-60 cells were divided into pcDNA3.1-NC, pcDNA3.1-LINC01268, si-NC, si-LINC01268, miR-NC, miR-217 mimics, si-LINC01268 + inhibitor-NC and si-LINC01268+ miR-217 inhibitor groups. The mRNA expressions of LINC01268 and miR-217 were detected by qRT-PCR. The targeting relationship between LINC01268 and miR-217 was detected by dual-luciferase reporter assay. Cell viability was detected by CCK-8 assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The expression of cell cycle and apoptosis-related proteins p21, Bcl-2, Bax, caspase-3 and PI3K/AKT signaling pathway-related proteins were detected by Western blot. The expression of LINC01268 in peripheral blood samples of AML patients and AML cell lines HL-60 and KG-1 was increased (P < 0.05), and the expression of miR-217 was decreased (P < 0.05). Compared with si-NC group and miR-NC group, the viability of HL-60 cells was decreased in si-LINC01268 group and miR-217 mimics group (P < 0.05), the proportion of cells in G1 phase and apoptosis rate were increased (P < 0.05), the protein expression levels of p21, Bax and caspase-3 were increased (P < 0.05), while the protein expression level of Bcl-2 was decreased (P < 0.05). LINC01268 targeted and negatively regulated the expression of miR-217, and inhibiting the expression of miR-217 partially reversed the effects of LINC01268 interference on the viability, cell cycle and apoptosis of HL-60 cells. Interference with LINC01268 could inhibit the activity of PI3K/AKT signaling pathway. Inhibiting the expression of miR-217 could partially reverse the inhibition of LINC01268 interference on PI3K/AKT signaling pathway. LINC01268 is highly expressed and miR-217 is lowly expressed in AML cells. LINC01268 can promote the activity of PI3K/AKT signaling pathway, increase the survival rate and inhibit the apoptosis of AML cells by targeting miR-217 expression.

Reduced Proliferation and Cell Death of Acute Myeloid Leukemia Cell Lines after Acalabrutinib Treatment

Background Acute myeloid leukemia (AML) still remains a significant challenge, with intensive chemotherapy treatments showing limited efficacy and lack of druggable targets in contrast to other hematologic malignancies. Exosomes, nanomolecules derived from cells reflect the parent cell's state, present opportunities for disease biomarker discovery and targeted therapy. Proteomic analysis of AML-derived exosomes may identify druggable targets despite limited prior research. In this study we investigated the potential of exosomes as a tool for target discovery in AML. Methods We conducted a proteomic analysis of AML-derived exosomes from HL-60, KG-1, and THP-1 cell lines, using fibroblast and mesenchymal stem cells as controls. Exosomes of each cell line were isolated, and digested via filter-aided sample preparation (FASP), followed by isobaric tagging using tandem mass tag (TMT) reagents. The samples were then fractionated using high-pH reversed-phase liquid chromatography and analyzed using Q-Exactive Orbitrap Hybrid Mass Spectrometry. Proteomic data were evaluated using SEQUEST in Proteome Discoverer 2.1 with a false discovery rate set to 1%. Differentially expressed proteins (DEPs) were identified and visualized in an interaction network and was analyzed for the gene ontology biological process (GOBP) using DAVID software. Protein-protein interactions were analyzed using the STRING software. Based on proteomic analysis, we identified the BTK-related pathway as potential druggable target for AML cells. AML cells were incubated with ACT, a BTK inhibitor, in various exposure time and density. Cell viability and proliferation rate was assessed using CCK-8 assay. BTK activity and signaling were assessed via Western blot analysis and immunocytochemistry of AML-derived exosomes. Cell death was evaluated using Annexin-V &amp; PI apoptosis kit and FACS analysis. Statistical analysis was performed to ascertain significance. Results Proteomics of exosomes from three AML cell types revealed distinct protein profiles via LC-MS, with 2450 unique proteins identified (Figure 1A). For KG1, HL60, and THP1, the number of up-regulated DEPs were 438, 434, and 408, and the number of down-regulated DEPs was 126, 118, and 89 (fold change&amp;gt;1.5 or &amp;lt;0.67, P&amp;lt;0.05). Gene ontology analysis identified four main categories of biological processes, including cell migration, T-cell pathways, cell damage, and vesicle formation pathways. Investigation into protein-protein interactions using the STRING software allowed selection of tyrosine kinases SYK and LYN as biomarker candidates. Both kinases regulate BTK, a critical molecule in B-cell antigen receptor signaling. SYK and LYN were found to be enriched in exosomes, promoting phosphorylation of BTK in recipient cells. (Figure 1B) Cell viability experiments with AML cell lines revealed reduced viability upon treatment with ACT. Viability decreased with increased exposure time and density, with the highest effects observed at 1,000nM ACT concentration for 48 hours (Figure 2A). This was accompanied by decreased cell numbers and proliferation rate which was significant in the HL-60 and KG-1 cell lines (Figure 2B). Western blot analysis demonstrated significant decrease of the BTK downstream signaling (P-BTK, PKC and p-ERK) by acalabrutinib treatment (1,000nM) at 48 hours (Figure 2C). Proliferative markers, such as PCNA, and Cyclin D1 were also significantly decreased. LYN and SYK was also decreased in the exosomes of HL-60 and THP-1 cell line. The inhibition of P-BTK and Cyclin D1 by ACT was further confirmed through Immunofluorescence assay. Finally, ACT was shown to promote cell death in AML cell lines, as evidenced by an apoptosis assay, with the most significant increases observed in cells treated exposed to 1,000nM ACT for 72 h (Figure 2D). Conclusion Overall, the findings of this study revealed that acalabrutinib reduced proliferation and induced cell death in AML cells suggesting the potential of BTK inhibitors in AML treatment. This was explained through proteomic analysis of AML-derived exosomes. However, further studies are required to validate the clinical use of exosomes as biomarkers and druggable target.

A Potential Link between hnRNP K and Cebpα: Implications for Ribosomal Dysfunction in Acute Myeloid Leukemia (AML)

Deletion of the long arm of chromosome 9, del(9q), is a recurrent genetic abnormality involving the haploinsufficient loss of the RNA binding protein hnRNPK and is often associated with poor treatment responses in AML. In addition to reduced hnRNP K expression, other critical proteins, not localized to this locus, are also aberrantly expressed in del9q AML. One protein of critical interest is CEBPα, a well-established myeloid differentiation transcription factor. Recently, CEBPα was discovered to have a novel role in ribosomal RNA (rRNA) synthesis. However, as opposed to CEBPα, hnRNP K's role in RNA stability, rRNA processing, and ribosomal functions is already firmly rooted. Collectively, these results suggest that del(9q) AML dysregulations in hnRNP K and CEBPα expression may have a multi-faceted influence on ribosomal biogenesis and protein translation. Previously, we used animal models and patient samples to demonstrate that Hnrnpk haploinsufficiency alters CEBPa expression, suggesting that reduced hnRNP K expression contributes to decreased CEBPα levels and activity. These findings led us to hypothesize that these distinct, yet interconnected proteins, play a cooperative role in regulating rRNA synthesis in two major ways. Firstly, hnRNP K directly governs rRNA processing and ribosomal loading. And secondly, via hnRNP K's mediated regulation of the nucleolar-localized isoform of CEBPα (p42 isoform), which was recently shown to enhance recruitment of RNA polymerase I to rRNA genes. To explore these relationships, we used AML patient samples, Hnrnpk haploinsufficient animal models, si- and sh- HNRNPK AML cell lines, and biochemical analyses. Initially, RPPA, CyTOF, and western blot analyses were used to investigate how reduced hnRNP K levels affect proteins involved in ribogenesis and protein translation. Here, we observed a direct correlation between hnRNP K expression and defects in proteins and pathways involved in protein translation, such as mTOR, phos-S6, and eEF- and eIF-family members, and CEBPa (log 10pAdj &amp;lt; .0001). Subsequently, we performed polysome assays that revealed 40S and 60S subunit formation was impaired in AML cell lines with reduced hnRNP K levels. Across multiple cell lines, reduced hnRNP K expression directly led to a significant increase in monosomes (unbound/inactive ribosomes; p &amp;lt; 0.01) and a significant decrease in polysome-bound RNA (p &amp;lt; 0.05) . Critically, HNRNPK haploinsufficiency and si- and sh- HNRNPK knock down experiments disrupted the abundance and processing of the 47S transcript (p &amp;lt; 0.05). To understand the impact that hnRNP K has on CEBPα in the context of rRNA biology, we performed co-immunoprecipitation of hnRNP K followed by RNA-seq (RIP-Seq) in human AML cell lines KG-1 and KG-1a. 1076 interacting RNAs were identified, which were strongly enriched in transcription factors implicated in myeloid biology, including CEBPA (log 2pAdj &amp;lt; .0001). Biochemical analyses of sequence-specific motifs conferred direct and functional interactions between hnRNP K and the 5‘ and 3‘ UTRs of the human CEBPA. These interactions support our in vivo findings that del9q AML and Hnrnpk haploinsufficiency lead to reduced CEBPA expression, specifically in the p42 isoform, potentially impacting its role in nucleolar localization and rRNA transcription regulation. Currently, we are evaluating the significance of these interactions at both the biological and translational levels. Using biochemical and cell-based assays, we are evaluating the direct effect that Hnrnpk haploinsufficiency has on rRNA abundance and on the processing of ITS products in the context of CEBPA. Through the use of viability screens, we are examining the efficacy of inhibitors of protein translation, translation initiation, and RNA Pol I on leukemic cells with 9q deletions of reduced hnRNP K expression. Altogether, these findings identify an uncharacterized relationship between del9qAML ( HNRNPK haploinsufficiency), CEBPα expression, and ribogenesis, providing valuable insight towards mechanisms driving this subtype of leukemia. Collectively, these data also suggest that del9q AML represents a novel and currently uncharacterized ribosomopathy.

Prognostic Significance of NCAM1 (CD56) Expression in AML Patients Treated with HMA+Venetoclax

Introduction : NCAM1 (CD56) is a cellular adhesion molecule highly expressed on natural killer (NK) cells and other immune cells. NCAM1 is aberrantly expressed in 20% of patients with acute myeloid leukemia (AML) and is associated with resistance to chemotherapy, upregulation of the MAPK pathway, and poor prognosis. The significance of NCAM1 in the setting of venetoclax (VEN) therapy has not been explored. We set out to determine the functional and prognostic role of CD56 in vitro and in newly diagnosed (ND) patients receiving VEN-based therapy. Methods : OCI-AML2, KG-1, and MOLM-14 cell lines were purchased from ATCC and maintained in RPMI, 10% or 20% FBS, L-Glutamine, and penicillin/streptomycin. Ex vivo drug testing on cell viability was performed using an MTS assay. Briefly, AML cells were plated for 72 hours in replicates of six at 1,250 cells/well in R20 and titrated using a log scale over 7 doses (0-1000 nM) of VEN. After 3 days of culture at 37 °C in 5% CO2, MTS reagent was added, optical density was measured at 490 nm, and raw absorbance values were adjusted to a positive control and (normalized to untreated wells to produce cell viability estimates). VEN-resistant (VR) OCI-AML2 cells were generated by continuous incubation with escalating doses of VEN. Viability was monitored by Guava EasyCyte. Cells maintained resistant by co-culture with 1 µM of VEN twice weekly. Flow for NCAM1 was performed with commercially available antibodies on parental and VR cells. NCAM1 expression on parental vs. VR OCI-AML2 cells was compared with student's T test. Inactivation of NCAM1 in OCI-AML2 cells was carried out by CRISPR/Cas9 using sgRNAs cloned into plentiCRISPRv2 (Addgene, #52961). AUC was derived from 4-parameter logistic regression curves fit to normalized cell viability values for the 7 tested drug doses. Clinical outcomes including overall survival (OS) were assessed in a retrospective cohort of ND AML patients treated with a hypomethylating agent (HMA)+VEN at Oregon Health &amp; Science University (OHSU). CD56 positivity on AML blasts was based on surface immunophenotype determined by multiparameter flow cytometry. OS was measured from AML diagnosis (with time from diagnosis to VEN start as left-truncation), estimated by Kaplan-Meier, compared with the log-rank test, and modeled with Cox regression to estimate unadjusted and adjusted hazard ratios (HRs) for CD56. Results : Across the 3 AML cell lines evaluated, higher baseline NCAM1 expression levels correlated with increased ex vivo resistance to VEN. The MOLM14 cell line was most resistant with highest median NCAM1 expression (90%), followed by OCI-AML2 with median NCAM1 expression (55%) and then KG-1 with lowest NCAM1 expression (3%). VR OCI-AML2 cells had higher expression of NCAM1 compared to parental OCI-AML2 cells (80% expression vs 55%; p=0.0003). CRISPR knockout of NCAM1 sensitized parental OCI-AML2 cells to VENcompared to cells containing a non-targeting control guide (Fig 1). To support our pre-clinical findings, we retrospectively identified N=98 ND AML patients treated with HMA+VEN at OHSU from 12/2018 to 12/2022. Median follow-up was 16 months and 60% of patients died. Median age at diagnosis was 73 (range=19-87), with 39% at least 75 years old. While 65% of patients were male, 63% had de novo AML and 23% had an antecedent hematologic disorder (AHD). By European LeukemiaNet (ELN) 2022 prognostic risk classification, 58% had adverse risk (14% unknown). The most common somatic mutations were IDH1/2 (27% of patients), NRAS/KRAS (26%), FLT3 ITD or TKD (19%), NPM1 (13%), and TP53 (13%). According to diagnostic flow, 30% of patients were CD56+, 22% CD11b+, 15% CD64+, and 85% HLA-DR+. Median (range) days between diagnosis and VEN start was 13 (1-285). NCAM1 positivity significantly correlated with shorter OS in the univariable (HR=1.80 [95% CI: 1.04-3.09], p=0.032; Fig 2) and multivariable settings (HR=2.10 [95% CI: 1.17-3.75], p=0.012, N=97), with the latter model controlling for age ≥75, AHD, and CD11b. Conclusion : NCAM1 expression is a marker of poor prognosis in ND AML patients treated with HMA+VEN. Knockout of NCAM1 in the OCI-AML2 cell line sensitized cells to VEN. Our data suggest that NCAM1 expression on AML blasts plays a biological role in promoting cellular resistance to VEN. Given emerging knowledge of VEN targeted downstream pathway associations, experiments are underway to explore the role of MAPK pathway and BCL2 family member dependence.

UBC9 Deficiency Accelerates AML Progression By Downregulating PARP1 Sumoylation and Subsequently Activating the NF-ΚB Pathway

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is a disease of heterogeneous clonal hematopoietic progenitor cells. Despite the relentless development of therapeutic options, including chemotherapy, and identification of many new therapeutic targets, the prognosis of AML is still poor, with a 5-year survival rate of approximately 35% in patients younger than 60 years old and 10% in patients older than 60 years, which may be due to the high degree of AML heterogeneity. Targeting the shared mechanisms of action among subclones has emerged led to find novel strategies, such as targeting protein post post-translational modification. Here we show that the impact of the UBC9 (the only E2 conjugation enzyme) and its related SUMOylation, a critical and reversible posttranslational modification as ubiquitination, on AML progression, to provide novel and potent sight for AML therapy. To identify the role of UBC9 expression in the clinical outcome of AML patients, we measured the expression of key SUMOylation genes, including SAE1, UBA2, UBC9, SUMO1, SUMO2, SUMO3, PIAS1, PIAS2, PIAS3, PIAS4, CBX4, and RANBP2, in the TCGA dataset (n=164) by using a multivariate survival curve based on the online tool SurvExpress. Our results showed that the survival probability curves of the low- and high-risk groups were significantly different due to the differences in the levels of expression of key SUMOylation genes ( p = 0.0011), and we found that the expression level of UBC9 was lower in the high-risk group than in the low-risk group. We next analyzed the UBC9 expression level and the prognosis of AML patients from the Therapeutically Applicable Research To Generate Effective Treatment dataset (n=156), which showed that AML patients with low UBC9 expression showed shorter overall survival ( p = 0.018)(Figure 1). In addition, real-time PCR showed that the UBC9 expression level in bone marrow samples from patients with de novo AML was significantly downregulated in both the high-risk patients based on National Comprehensive Cancer Network guidelines and the liver or spleen enlargement patients ( p = 0.01 and p = 0.001, respectively) (Figure 1). We used RNA interference system to knock out Ubc9 in a mouse model of MLL-AF9 and MLL-ENL induced AML. We observed the survival of AML mice and found that Ubc9 fl/+;Vav-Cre recipient mice showed a significantly shorter survival time than Ubc9 fl/+ mice (Figure 2). In addition, apoptosis experiments revealed reduced apoptosis of leukemia cells and leukemia stem cells in Ubc9 fl/+;Vav-cre mice. HE staining showed significantly higher leukemia cell infiltration in the livers, meninges, and kidneys of Ubc9 fl/+;Vav-cre mice compared to Ubc9 fl/+ mice. Transwell experiments indicated that Ubc9 deficiency resulted in increased migration of leukemia cells. Colony forming assay demonstrated that Ubc9-deficient leukemia cells formed significantly larger colonies. Flow cytometry analysis showed that Ubc9-deficient leukemia cells expressed high levels of c-kit and low levels of Mac1. To further elucidate the role of UBC9 in human AML cells, we constructed UBC9 deficient KG1 and K562 cell lines by using CRISPR-Cas9 technology. The UBC9 KD cell lines presented the same phenotype such as the increase of cell viability and the enhancement of invasiveness. To better understand the role that UBC9 plays in AML cell biology, we performed a system-level approach based on quantitative SUMO proteomic analysis to identify potential UBC9 substrates and then identified PARP1 with SUMOylation site lysine residue 486 (K486) by multiomics analysis. Finally, we validated that UBC9-mediated PARP1 SUMOylation played an important role in AML progression by activating the NF-kappa B pathway via the site-directed mutation of PARP1 (K486R) in UBC9-KO AML cells by using western blot and Co-immunoprecipitation. Together, our study revealed that UBC9 KO accelerated AML progression and enabled us to contrast global SUMOylation profiles of UBC9 in AML cells. In addition, we found that the PARP1 SUMOylation-mediated NF-κB pathway activation is a positive regulatory mechanism in the tumorigenesis of AML. These findings may provide potential clinical implications for AML treatment.

Antitumor Activity of Ziziphus Jujube Fruit Extracts in KG-1 and NALM-6 Acute Leukemia Cell Lines

Background: Ziziphus jujube Mill. belongs to the Rhamnaceae family. It has been reported to have a variety of biological activities such as antitumor, antioxidant, and anti-inflammatory effects. This study investigates the antiproliferative effect of Ziziphus jujube on KG-1 and NALM-6 acute leukemia cell lines.&#x0D; Materials and Methods: In this experimental study, the aqueous, ethyl acetate, and hydroalcoholic extracts of the Ziziphus jujube were prepared. Total phenolic and flavonoid components were detected because the presence of these compounds is associated with antioxidant and anticancer effects. Different concentrations of extracts were prepared, and KG-1 and NALM-6 cell lines were treated with them at 12, 24, 36, and 48 hours. Cell viability and IC50 values of the extracts were calculated using MTT assays. BD Cycle TEST PLUS DNA Kit was used for cell cycle progression analysis. Bcl2, Bax, and caspase-3 mRNA expressions were also assessed.&#x0D; Results: Cell viability decreased in a concentration-dependent manner. The best efficacy belonged to the ethyl acetate extract. Investigation of cell cycle progression demonstrated that the number of G0/G1 cells enhanced and the number of G2/M cells decreased when the ethyl acetate extract was applied in its IC50 concentration. A considerable increase in Caspase-3 and Bax and a decrease in Bcl2 gene expression were detected in molecular examination.&#x0D; Conclusion: According to our research, Ziziphus jujube ethyl acetate extract has antitumor properties on KG-1 and NALM-6 cell lines, possibly through induction of apoptosis and cell cycle regulation.

Human Umbilical Cord Mesenchymal Stem Cell-Derived Microvesicles Could Induce Apoptosis and Autophagy in Acute Myeloid Leukemia.

Microvesicles (MV) have been identified as candidate biomarkers for treating acute myeloid leukemia (AML). This study investigated the effects of human umbilical cord-derived mesenchymal stem cell (hUCMSC)-derived MVs on apoptosis and autophagy in the KG-1 leukemic cell line. The hUCMSCs were cultured and characterized by flow cytometry. MVs were isolated by ultracentrifugation, and the concentration was determined using the Bradford method. The characteristics of MVs were confirmed using transmission electron microscopy, flow cytometry, and dynamic light scattering methods. KG-1 cells were treated with the desired concentrations of MVs for 24 h. The apoptosis induction and reactive oxygen species production were evaluated using flow cytometry. RT-PCR was performed to evaluate apoptosis- and autophagy-related genes expression. Following tretment of KG-1 cells with 25, 50, and 100 μg/ml concentrations of MVs, the apoptosis rates were 47.85%, 47.15%, and 51.35% (p < 0.0001), and the autophagy-induced ROS levels were 73.9% (p < 0.0002), 84.8% (p < 0.0001), and 85.4% (p < 0.0001), respectively. BAX and ATG7 gene expression increased significantly at all concentrations compared to the control, and this level was higher at 50 μg/ml than that of the other concentrations. In addition, LC3 and Beclin 1 expression increased significantly in a concentration-dependen manner. Conversely, BCL2 expression decreased compared to the control. Our findings indicate that hUCMSC-MVs could induce cell death pathways of autophagy and apoptosis in the KG-1 cell lines and exert potent antiproliferative and proapoptotic effects on KG-1 cells in vitro. Therefore, hUCMSC-MVs may be a potential approach for cancer therapy as a novel cell-to-cell communication strategy.

Examination of in vitro and in vivo antitumor activity of Menin-MLL inhibitor, D0060-319.

e15119 Background: Rearrangements of the mixed-lineage leukemia (MLL) gene result in the production of MLL fusion proteins, which occurs in 5-10% of acute leukemia in adults and &gt; 70% of infant leukemia, respectively. Menin protein, encoded by the MEN1 gene, interacts with MLL fusion proteins to induce leukemia. D0060-319, a Menin-MLL inhibitor, shows a significant antitumor effect in vivo and in vitro. Methods: The fluorescence polarization (FP) binding assay to assess inhibition of D0060-319 to Menin-MLL interaction. The cross reactivity of 44 molecular targets was tested to assess their off-target binding potential. The anti-proliferative ability of D0060-319 was evaluated by CCK-8 in different cell lines. The in vivo anti-tumor effect of D0060-319 was evaluated in a human MV4-11 and MOLM-13 cell mouse xenograft tumor model. Briefly, five million MV4-11 cells or 500 thousand MOLM-13 cells were subcutaneously implanted the right flank of CB-17 SCID mice to establish cell mouse xenograft tumor model. When tumor volume reached 100-120mm3, the animals were grouped and administrated with D0060-319 at the dose of 25, 50, 75, 100 mg/kg or vehicle at twice daily for 21days. Tumor measurements and body weights were performed twice weekly to assess the antitumor efficacy and tolerability of D0060-319. Results: D0060-319 competitively binds Menin to inhibit the interaction with MLL fusion protein with IC50 of 7.46nM, has high antiproliferative activity in MV4-11 and MOLM-13 cell lines containing MLL fusion proteins with IC50 values of 4.0nM and 1.7nM, respectively. However, in Kasumi-1, K562, HL-60 and KG-1 cell lines lacking MLL rearrangement, D0060-319 antiproliferative activity with IC50 &gt; 10 μM, indicating selectivity for leukemia cell lines containing MLL fusion proteins active. In the MV4-11 cell mouse xenograft tumor model, D0060-319 significantly inhibited tumor growth at the dose of 25mg/kg, with a tumor growth inhibition (TGI) rate of 82.97%, and other doses (50, 75 and 100mg/kg) tumor completely regressed. In the MOLM-13 cell mouse xenograft tumor model, a dose-dependent anti-tumor effect was observed after 14 days of administration, with TGIs of 81.08%, 88.47% and 90.26% at the dose of 50, 75 or 100 mg/kg, respectively. In both CDX models, no bodyweight loss was observed, suggesting that D0060-319 was well tolerated. Furthermore, off-target screening assays showed that D0060-319 did not cross-react with any of the 44 molecular targets at 10 μM, indicating that D0060-319 is highly selective, high-affinity for the MLL-binding pocket on Menin. Conclusions: In MLL-rearranged leukemia, D0060-319 showed high antitumor activity and good tolerability both in vitro and in vivo.

Abstract A32: Exemestane inhibits survival and cell cycle regulators and retards AML cell proliferation

Abstract Acute myeloid leukemia (AML) is the most common type of leukemia with poor prognosis. Although estrogen plays important roles in female cancers, the recent reports indicate that estrogen and its receptors also attribute to the development of non-gender cancers and AML is one of those. Therefore, the inhibition of estrogen production becomes a potential strategy for AML treatment. Exemestane is an aromatase inhibitor and commonly used for reducing estrogen production in breast cancer patients. Our data showed that Exemestane treatment significantly decreased the proliferation of both KG-1 and HL-60 AML cell lines. Since STAT3 and AKT closely collaborate with estrogen receptor in breast cancer cells, their activations are next to be monitored. In KG-1 cells, the phosphorylations of STAT3 (Y705 and S727) and AKT (S473) were both inhibited by Exemestane treatment. Regarding the cell cycle control, p21 was increased by Exemestane in KG-1 cells in which Cyclin E was reduced. It suggests that CDK2 might be inactivated by Exemestane-reduced Cyclin E and therefore p21 escaped from the CDK2-dependent degradation. Although the signaling findings of Exemestane in KG-1 were interesting, no similar results were found in HL-60 cells except the growth inhibition, suggesting the distinct regulation mechanisms between two cell lines. In summary, these results not only suggest, again, the importance of estrogen demand for AML cells, but also show the novel and potential treatment of AML using blockade of estrogen supply, which could be helpful to uncover novel AML treatments in the future. Citation Format: Mei-chih Chen, Pang-Ting Cheng, Yu-Chiao Cheng, Ho Lin, Chieh-Lin Jerry Teng. Exemestane inhibits survival and cell cycle regulators and retards AML cell proliferation [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A32.

Shotgun Lipidomics Elucidates the Lipidome Alterations of the Mcl-1 Inhibitor S63845 in AML Cell Lines with a Focus on Sphingolipids

Objective: Acute myeloid leukemia (AML) is a vigorous type of leukemia requiring effective treatment. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic molecule that is upregulated in AML and is studied as a target for treatment. The specific Mcl-1 inhibitor, S63845, has antiproliferative effects on AML cells. Bioactive sphingolipids have crucial roles in cells and regulate Mcl-1 stability. This study aimed to elucidate the changes in lipid profiles of AML cell lines in response to Mcl-1 inhibitor S63845 treatment, with a special focus on sphingolipids. Materials and Methods: The cytotoxic effects of S63845 were identified in the AML cell lines MV4-11, HL60, and KG1 using the MTT cell proliferation assay. Lipidome analysis was conducted by quantitative shotgun lipidomics covering 378 individual lipid species in 26 classes within the major lipid categories. Results: The IC50 values of S63845 have been calculated as 7 nM for MV4-11, 53 nM for HL60, and 479 nM for KG1. The lipidome results reveal the S63845 treatment to increase ceramide (Cer) levels in the MV4-11 and KG1 cell lines at the expense of downstream sphingolipids while increasing the hexosylceramide (HexCer) levels in the HL60 cell line at the expense of the Cer and sphingomyelin (SM). Conclusion: This study showed S63845 to be able to suppress cell proliferation by altering lipid compositions in AML cell lines. More importantly, the study suggested S63845 to differentially affect the lipid profiles of AML cell lines.

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

A pyridinesulfonamide derivative FD268 suppresses cell proliferation and induces apoptosis via inhibiting PI3K pathway in acute myeloid leukemia.

Aberration of PI3K signaling pathway has been confirmed to be associated with several hematological malignancies including acute myeloid leukemia (AML). FD268, a pyridinesulfonamide derivative characterized by the conjugation of 7-azaindole group, is a newly identified PI3K inhibitor showing high potent enzyme activity at nanomole concentration. In this study, we demonstrated that FD268 dose-dependently inhibits survival of AML cells with the efficacy superior to that of PI-103 (pan-PI3K inhibitor) and CAL-101 (selective PI3Kδ inhibitor) in the tested HL-60, MOLM-16, Mv-4-11, EOL-1 and KG-1 cell lines. Further mechanistic studies focused on HL-60 revealed that FD268 significantly inhibits the PI3K/Akt/mTOR signaling pathway, promotes the activation of pro-apoptotic protein Bad and downregulates the expression of anti-apoptotic protein Mcl-1, thus suppressing the cell proliferation and inducing caspase-3-dependent apoptosis. The bioinformatics analysis of the transcriptome sequencing data also indicated a potential involvement of the PI3K/Akt/mTOR pathway. These studies indicated that FD268 possesses high potent activity toward AML cells via inhibition of PI3K/Akt/mTOR signaling pathway, which sheds some light on the pyridinesulfonamide scaffold for further optimization and investigation.

Structure-activity relationship analysis of novel GSPT1 degraders based on benzotriazinone scaffold and its antitumor effect on xenograft mouse model

Molecular glue degraders, such as lenalidomide and pomalidomide, bind to cereblon (CRBN) E3 ligase and subsequently recruit neosubstrate proteins, Ikaros (IKZF1) and Aiolos (IKZF3), for the ubiquitination-proteasomal degradation process. In this study, we explored structure–activity relationship analysis for novel GSPT1 degraders utilizing a benzotriazinone scaffold previously discovered as a novel CRBN binder. In particular, we focused on the position of the ureido group on the benzotriazinone scaffold, substituent effect on the phenylureido group, and methyl substitution on the benzylic position of benzotriazinone. As a result, we identified 34f (TD-522), which exhibits strong anti-proliferative effects in both KG-1 (EC50 = 0.5 nM) and TMD-8 (EC50 = 5.2 nM) cell lines. Compound 34f effectively induced GSPT1 degradation with a DC50 of 0.269 nM and Dmax of >95 % at 10 nM concentration in KG-1 cells. An in vivo xenograft study showed that compound 34f effectively suppressed TMD8-driven tumor growth, suggesting a potential role in the development of novel GSPT1 degraders.

Native Endophytes of Tripterygium wilfordii-Mediated Biotransformation Reduces Toxicity of Celastrol

Celastrol (1), obtained from the roots of Tripterygium wilfordii Hook F., is most likely to become an antitumor drug, but with severe cytotoxicity. Due to the lack of modifiable sites in the structure of celastrol, the structural diversity of the modified products obtained by synthesis in the previous studies is insufficient, which hinders the pace of its patent medicine. This study describes a method of microbial transformation to increase the modification site of celastrol and reduce its toxicity. The screening of endophytes from native plants was introduced in this context, which led to two novel stereoselective oxidation products such as S-16-hydroxyl celastrol (2) and A-ring aromatized S-16-hydroxyl celastrol (3), along with a rare 7,9-octadecadienoic acid ester of celastrol (4). Their structures were determined by extensive spectroscopic data analysis, especially 1D and 2D NMR. Compared with 1, compounds 3 and 4 exhibited similar antitumor activity in U251, A549, KG-1, and B16 cell lines. Compound 2 had slightly decreased antitumor activity when compared with compound 1. Furthermore, compound 2–4 showed lower cytotoxicity against BV-2 (about 21-fold lower, 2: 92.82 μM, 3: 34.25 μM, and 4: 74.75 μM vs. celastrol: 4.35 μM), and also identical trends against H9c2 and PC12 cell lines.