Lymphoblastic Leukemia Cell Line Research Articles

Elevated Bax/Bcl-2 Ratio: A Cytotoxic Mode of Action of Kermanian Propolis Against an Acute Lymphoblastic Leukemia Cell Line, NALM-6.

Currently, alternative cancer remedies, especially herbal-derived medicines, have attracted great interest. Propolis, a honeybee-produced naturopathic formulation, is an available, affordable, and safe example of such remedies with different content according to its geographic location. Findings regarding the protective properties of this resinous substance across numerous pathological conditions are promising. Although the anti-tumor effects of propolis from different origins have been explored to some degree, yet there is no study on the effects of Kermanian propolis in the treatment of hematologic malignancies. Accordingly, the objective of the present experiment was to divulge the anti-tumor potential of this bioactive substance both as monotherapy and in combination with doxorubicin against an acute lymphoblastic leukemia cell line (NALM-6).The viability of cells treated with Kermanian propolis (5-500μg/mL) and doxorubicin (5-100μg/mL) was analyzed during 72h. Based on the MTT results, the best incubation time, IC50 concentrations, and finally the cytotoxicity of the combination therapy were ascertained. Next, the apoptotic rate and expression of apoptosis-related genes (Bcl-2 and Bax) were assessed in mono and combination therapies using flow cytometry and real-time PCR assays, respectively. Kermanian propolis and doxorubicin have impressive tumor-suppressing activity in a dose-dependent manner (IC50 concentrations: 100 and 40μg/mL respectively). The best incubation time was considered 48h. For the combination approach, 50 and 10μg/mL were determined as optimum concentrations of the compounds. The selected concentrations induced notable apoptosis in the studied cells through significant (P < 0.01) upregulation of Bax/Bcl-2 level. The present study clearly suggests that Kermanian propolis, as an adjunct treatment option, has a promising apoptosis-induced cell death potential in the NALM-6 cell line.

Changes in intracellular activation-related gene expression and induction of Akt contribute to acquired resistance toward nelarabine in CCRF-CEM cell line

Drug resistance is a major problem in treatment with nelarabine, and its resolution requires elucidation of the underlying mechanisms. We established two nelarabine-resistant subclones of the human T-cell lymphoblastic leukemia cell line CCRF-CEM. The resistant subclones showed changes in the expression of several genes related to nelarabine intracellular activation and inhibition of apoptosis. Activation of the Akt protein upon nelarabine treatment was observed in both subclones. The combination treatment with nelarabine and PI3K/Akt inhibitors was shown to inhibit cell growth. Cross-resistance was observed with ara-C and not with vincristine, daunorubicin, or etoposide treatment. Thus, changes in the expression of cellular activation-related genes, inhibition of apoptosis, and induction of Akt may be involved in the development of nelarabine resistance in the CCRF-CEM cell model. The use of different classes of chemotherapeutic agents and combination therapy with PI3K/Akt pathway inhibitors may be used to overcome resistance to nelarabine.

A Novel Compound Plumercine from Plumeria alba Exhibits Promising Anti-Leukemic Efficacies against B Cell Acute Lymphoblastic Leukemia

The current study was focused to evaluate the antioxidant and the anti-cancerous properties of Plumeria alba. Plumeria alba was chosen due to its existing medicinal values. Antioxidant assays like Superoxide radical scavenging assay, Nitrous Oxide radical scavenging assay, were performed, on the methanolic and ethyl acetate extracts of the plant, that depicts the pro-oxidant nature of the extract. Further, they were tested to check cell viability on B cell Acute Lymphoblastic Leukemia (ALL) Cell line (NALM 6), human lung cancer cell line (A549), T cell Acute Lymphoblastic Leukemia cell line (MOLT4), and PBMC isolated from normal donors utilizing MTT assay. Robust anti-proliferative activity was observed in the case of NALM 6 followed by A549, MOLT4, whereas negligible activity was observed in the case of PBMC. Intrigued by this finding, in silico docking was performed using three bioactive compounds namely Plumericine, Isoplumericine, and 13-O-p-Coumaroylplumieride, unique to Plumeria sp. They were docked against five different cyclins and Cdk proteins responsible for ALL. The compounds have shown satisfactory results and their druggability and ADMET properties were checked further. Plumercine turned out to be the most competent compound and hence can be considered as a potential leukemic drug candidate in the future.

Formulation, Characterization, Antioxidant, Cytotoxicity, and Anti-acute Leukemia Effects of Fe Nanoparticles.

In this study, iron nanoparticles were prepared and synthesized in aqueous medium using Cinnamomum verum as stabilizing and reducing agents. We determined the anti-acute leukemia potentials of FeNPs against acute T cell leukemia and acute lymphoblastic leukemia cell lines. FeNPs inhibited half of the DPPH molecules in the concentration of 139 µg/mL. MTT assay was used on J.RT3-T3.5 (Acute T cell leukemia cell line), Jurkat, Clone E6-1 (Acute T cell leukemia cell line), MOLT-3 (Acute lymphoblastic leukemia cell line), TALL-104 (Acute lymphoblastic leukemia cell line), and HUVEC (Normal cell line) for analyzing of cytotoxicity and anti-acute leukemia effects of FeNPs. These nanoparticles had high cell death and anti-acute leukemia effects against J.RT3-T3.5, Jurkat, Clone E6-1, MOLT-3, and TALL-104 cell lines. Among the above cell lines, the best result of anti-acute leukemia properties of composite was gained in the cell line of Jurkat, Clone E6-1. All result showed the iron nanoparticles may be used as a chemotherapeutic treatment drug of leukemia in humans.

More Related Gene Pathways to Vincristine-Induced Death Events in a Human T-Acute Lymphoblastic Leukemia Cell Line.

Acute lymphoblastic leukemia (ALL) is common in children but rare in adults. Vincristine (VCR) is one of the drugs used at the beginning of treatment. Some genes are resistant to VCR in B-ALL. Here, we examined the effect of VCR on gene expression changes in a T-ALL cell line, Jurkat. The MTT method was used to determine the IC50 in Jurkat cells treated with different concentrations of VCR for 48 and 72 hours. Total RNA was isolated from the cells and cDNA was prepared. The Human Cancer Drug Target PCR Array kit was used to evaluate the 84 gene expression changes in Jurkat cells. Protein-protein interaction was analyzed by STRING software. We identified 66 differentially expressed genes as comparison to untreated cells. The response to VCR-induced apoptotic events was remarkable in the pathways of heat shock protein, topoisomerases, protein kinases, cathepsins and cell cycle. In other pathways, there were resistant genes as well as sensitive genes to VCR treatment. Some proteins like HSP90AA1 and ESR1 had determining associations with other proteins. The results suggest VCR target genes in T-ALL cells may be beneficial biomarkers for ALL treatment and can be used to select appropriate synergistic drugs for VCR.

Poincaré Maps and Aperiodic Oscillations in Leukemic Cell Proliferation Reveal Chaotic Dynamics.

Biological systems are dynamic systems featuring two very common characteristics; Initial conditions and progression over time. Conceptualizing this on tumour models it can lead to important conclusions about disease progression, as well as the disease’s “starting point”. In the present study we tried to answer two questions: (a) which are the evolving properties of proliferating tumour cells that started from different initial conditions and (b) we have attempted to prove that cell proliferation follows chaotic orbits and it can be described by the use of Poincaré maps. As a model we have used the acute lymphoblastic leukemia cell line CCRF-CEM. Measurements of cell population were taken at certain time points every 24 h or 48 h. In addition to the population measurements flow cytometry studies have been conducted in order to examine the apoptotic and necrotic rate of the system and also the DNA content of the cells as they progress through. The cells exhibited a proliferation rate of nonlinear nature with aperiodic oscillatory behavior. In addition to that, the (positive) Lyapunov indices and the Poincaré representations in phase-space that we performed confirmed the presence of chaotic orbits. Several studies have dealt with the complex dynamic behaviour of animal populations, but few with cellular systems. This type of approach could prove useful towards the understanding of leukemia dynamics, with particular interest in the understanding of leukemia onset and progression.

Describing a novel chemotherapeutic drug formulated by Diosmin for the treatment of acute lymphoblastic leukemia and diabetes mellitus

IntroductionDiosmin is a natural citrus flavone with remarkable antioxidant and anti-inflammatory features. Acute leukemia is a common type of cancer that is caused in the blood.Material and methodsAlpha amylase activity was determined by a method adapted from the work of Taha et al.ResultsIn this study, we examined its effect on some important enzymes, the IC50 values were 196.07 for Aldose reductase, and 76.40 for alpha amylase. The molecular docking study was performed to assess the binding affinity and biological activities of diosmin in the presence of alpha amylase and aldose reductase. The results of the docking study indicated that diosmin has a remarkable binding affinity to these enzymes with a docking score of -9.768 and -140469 for alpha-amylase and aldose reductase, respectively. Therefore, this compound could be used as a potential inhibitor for these enzymes. In the cellular and molecular part of the recent study, the treated cells with Diosmin were assessed by MTT assay for 48h about the cytotoxicity and anti-human acute lymphoblastic leukemia properties on HL-60, Clone 15 HL-60, HL-60/MX1, and HL-60/MX2 cell lines. The IC50 of Diosmin were 466, 323, 502, and 537 µg/mL against HL-60, Clone 15 HL-60, HL-60/MX1, and HL-60/MX2 cell lines, respectively.ConclusionsThe viability of acute lymphoblastic leukemia cell lines reduced dose-dependently in the presence of Diosmin. It appears that the anti-human acute lymphoblastic leukemia effect of Diosmin is due to their antioxidant effects.

Drug resistance to nelarabine in leukemia cell lines might be caused by reduced expression of deoxycytidine kinase through epigenetic mechanisms.

Drug resistance is a serious problem in leukemia therapy. A novel purine nucleoside analogue, nelarabine, is available for the treatment of children with T cell acute lymphoblastic leukemia. We investigated the mechanisms of drug resistance to nelarabine. Nelarabine-resistant cells were selected by stepwise and continuous exposure to nelarabine using the limiting dilution method in human B and T cell lymphoblastic leukemia cell lines. Expression analysis was performed using real-time polymerase chain reaction, and epigenetic analysis was performed using methylation-specific polymerase chain reaction and chromatin immunoprecipitation. The RNA expression level for deoxycytidine kinase (dCK) was decreased in nelarabine-resistant leukemia cells. There were no differences between the parental and nelarabine-resistant leukemia cells in the methylation status of the promoter region of the dCK gene. In the chromatin immune precipitation assay, decreased acetylation of histones H3 and H4 bound to the dCK promoter was seen in the nelarabine-resistant cells when compared to the parental cells. Furthermore, treatment with a novel histone deacetylase inhibitor, vorinostat, promoted the cytotoxic effect of nelarabine along with increased expression of the dCK gene, and it increased acetylation of both histones H3 and H4 bound to the dCK promoter in nelarabine-resistant leukemia cells. The combination index showed that the effect of nelarabine and vorinostat was synergistic. This study reports that nelarabine with vorinostat can promote cytotoxicity in nelarabine-resistant leukemia cells through epigenetic mechanisms.

Characterization of a novel glucocorticoid-resistant human B-cell acute lymphoblastic leukemia cell line, with AMPK, mTOR and fatty acid synthesis pathway inhibition

BackgroundAcquired glucocorticoid (GC) resistance remains the main obstacle in acute lymphoblastic leukemia (ALL) therapy. The aim of the present study was to establish a novel GC-resistant B-ALL cell line and investigate its biological characteristics.MethodsA cell culture technique was used to establish the GC-resistant cell line from the parental cell, NALM-6. Molecular and cellular biological techniques including flow cytometry, MTT assay, western blotting, DNA fingerprinting analysis and whole transcriptome sequencing (WTS) were used to characterize the GC-resistant cell lines. Nude mice were used for xenograft studies.ResultsThe GC-resistant cell line, NALM-6/HDR, was established by culturing NALM-6 cells under hypoxia for 5 weeks with a single dexamethasone (Dex) treatment. We subcloned the NALM-6/HDR cell lines, and got 6 monoclone Dex-resistant cell lines, NALM-6/HDR-C1, C3, C4, C5, C6 and C9 with resistance index (RI) ranging from 20,000–50,000. NALM-6/HDR and its monoclone cell line, NALM-6/HDR-C5, exhibited moderate (RI 5–15) to high resistance (RI > 20) to Ara-c; low or no cross-resistance to L-Asp, VCR, DNR, and MTX (RI < 5). STR analysis confirmed that NALM-6/HDR and NALM-6/H were all derived from NALM-6. All these cells derived from NALM-6 showed similar morphology, growth curves, immunophenotype, chromosomal karyotype and tumorigenicity. WTS analysis revealed that the main metabolic differences between NALM-6 or NALM-6/H (GC-sensitive) and NALM-6/HDR (GC-resistant) were lipid and carbohydrates metabolism. Western blotting analysis showed that NALM-6/HDR cells had a low expression of GR and p-GR. Moreover, AMPK, mTORC1, glycolysis and de novo fatty acid synthesis (FAS) pathway were inhibited in NALM-6/HDR when compared with NALM-6.ConclusionsNALM-6/HDR cell line may represent a subtype of B-ALL cells in patients who acquired GC and Ara-c resistance during the treatment. These patients may get little benefit from the available therapy target of AMPK, mTORC1, glycolysis and FAS pathway.

Preclinical Results of an Allogeneic, Universal CD19/CD7-Targeting CAR-T Cell Therapy (GC502) for B Cell Malignancies

BackgroundAutologous CD19 CAR-T therapies show very promising clinical efficacy, but are limited in their applicability by several factors including cost, time to manufacture, and other factors involving patients own T-cell qualities. GC027, a CD7 targeting allogeneic, universal CAR-T (UCAR-T) currently in development for the treatment of T-cell acute lymphoblastic leukemia (T-ALL) has demonstrated robust expansion and anti-leukemia efficacy with a manageable safety profile in an investigator-initiated trial in China. These data suggest that, a single CD7 targeting CAR-T therapy is able to generate a therapeutic window by suppressing host vs graft (HvG) rejection of UCAR-T cells by patients' own NK and T cells, and achieve efficacy in patients with T-ALL. Based on these findings we developed GC502, a CD19/CD7 dual-targeting, allogeneic CAR-T therapy for B-cell malignancies, in which the CD19 CAR moiety targets malignant cells while CD7 CAR moiety suppresses HvG in variety of preclinical models.MethodsGC502 was manufactured using leukopaks from HLA-unmatched healthy donors. It contains a 4-1BB based 2 nd-generation dual targeting CAR comprising an anti-CD19 and an anti-CD7 single-chain variable fragments (scFvs). TRAC and CD7 loci were disrupted to avoid graft vs host disease and fratricide, respectively. To select the leading CAR candidate, CAR expression and functionalities of CAR constructs with different heavy-light (H-L) chain orientations of the dual CAR were analyzed via in vitro assays and mouse xenograft models, in comparing to single CD19 CAR and CD7 CAR products. To achieve optimal anti-tumor efficacy, a T-cell enhancer was included in the CAR construct.ResultGene editing and dual CAR orientation selectionTRAC and CD7 double knockout efficiencies were constantly above 97% across multiple donor pan T cells. Although CD19/CD7 CAR expression levels in different H-L chain orientations were similar, in the final CAR-T product as measured by flow cytometry (FCM) analysis, significant difference was observed in their cytotoxicity and in vitro expansion under repeated antigen stimulations by CD19+ B-cell acute lymphoblastic leukemia (B-ALL) cell line Nalm6 and CD7+ T-cell line CCRF-CEM. CAR candidates mediated the strongest cytotoxicity and most durable response were selected for further optimizations.CAR construct optimizationsFor the leading candidates, we first assessed the dual CAR efficacy after incorporation of an enhancer. While the IL-2, TNFα and IFNγ secretion levels were comparable, enhancer addition significantly improved tumor killing and CAR-T cell expansion under repeated stimulations by either CD19+ or CD7+ target cells. Anti-leukemia response under sub-optimal CAR-T cell dosages were also greatly enhanced as assessed by both B-ALL and T-ALL mouse xenograft models.GC502 CAR functionality comparison to single CAR products with proven clinical efficaciesGC502 and GC027 were compared for their CD7 CAR function to assess their anti-HvG activities. GC502 and GC027 exhibited comparable toxicities towards pan T cells and similar efficacies in a highly malignant T-ALL mouse model. The CD19 CAR functionality of GC502 were evaluated and compared to a 2 nd generation CD19 CAR product comprising a FMC63 scFv and a 4-1BB-CD3ȥ signaling domain. In a Raji based B-ALL mouse xenograft model, both products rapidly eliminated cancer cells. While CD19 CAR treated mice showed signs of relapse at 2 weeks post CAR-T infusion, GC502 treatment group maintained “leukemia free” status till the end of study (Day28).ConclusionGC502 was optimized for CD19/CD7 dual CAR functionality and in vivo durability. It demonstrated robust anti-tumor efficacy and promising potentials to suppress HvG. This report presents an example that the dual CAR design of GC502 may serve as a novel “off-the-shelf” CAR-T technology. DisclosuresGe: GracellBiotechnologies Ltd: Current Employment, Current equity holder in publicly-traded company. Yang: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Sun: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Chen: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Qiu: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Yin: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Shen: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Sersch: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Cao: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Wang: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company.

Inhibition of Mitochondrial Translation By the Marine Natural Product Elatol Shows Potent Antileukemia Activity

Targeted signaling inhibitors for hematologic malignancies may lead to limited clinical efficacy due to the outgrowth of subpopulations with alternative pathways independent of the drug target. Relapse/refractory disease that results from treatment with targeted signaling inhibitors is a major hurdle in obtaining curative responses. Interestingly, work over the past decade or more has shown that chronic myelogenous leukemia (CML) stem cells (CD34+CD38-) are resistant to targeted signaling inhibitors, such as the BCR-ABL kinase class of inhibitors, often a problematic source of resistance leading to residual disease that may precipitate later progression (Hamilton et al., 2012).Recent studies have shown that some forms of lymphoma and leukemia cell have an energy metabolism highly dependent on mitochondrial oxidative phosphorylation (Ashton et al., 2018). Tigecycline, a US FDA approved antibiotic, has been shown to inhibit synthesis of mitochondrion-encoded proteins due to the similarity of bacterial and mitochondrial ribosomes, leading to selective lethality in hematologic malignancies reliant on enhanced oxidative phosphorylation (Norberg et al., 2017). Indeed, it was established that CML stem cells are reliant on upregulated oxidative phosphorylation, and combination treatment with the tyrosine-kinase inhibitor (TKI) imatinib and tigecycline eradicated therapy-resistant CML, both in vitro and in animal models (Kuntz et al., 2017).We have previously reported that elatol, the major compound from the red alga Laurencia microcladia, is effective against several non-Hodgkin lymphomas and primary chronic myelogenous leukemia cells (Peters et al., 2018). In vitro studies showed that elatol inhibits eIF4A1 helicase activity, suppressing cytoplasmic cap-dependent translation initiation. Further assessments using 35-S-methionine incorporation in HEK293T cells with or without single-digit micromolar concentrations of elatol for short time periods revealed strong downregulation of mitochondrion-encoded proteins as in Figure 1, (with no effect on mitochondrial transcription). This was confirmed in CML and acute lymphoblastic leukemia (ALL) cell lines whose 24-hour elatol LD50 ranged from high nanomolar to low micromolar concentrations. This potency was 10-40x higher than for tigecycline in side-by-side comparisons across several leukemia cell lines when compared at 72h. Additionally, we established that elatol does not affect integrity of small and large mitochondrial ribosomal units through sedimentation property analysis using sucrose gradients. Although the specific target on the mitochondrial translation apparatus remains elusive, we have uncovered that its mechanism of action differs from that of chloramphenicol, which inhibits translation elongation.In summary, we have performed proof-of-concept studies using HEK293T and HeLa cell lines, isolated mitochondria from HEK293T, and CML and ALL cell lines to reveal that elatol is a potent inhibitor of mitochondrial protein synthesis at concentrations that do not affect cytoplasmic protein synthesis and that this mechanism differs from chloramphenicol. Tigecycline's compelling preclinical data in combination with TKI informed design of a pending clinical trial (NCT02883036). Elatol's greatly improved potency provide a potential starting point for further optimization of this paradigm. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Redefining Hyperviscosity in Acute Leukemia: Implications for Red Cell Transfusions

Hyperleukocytosis, most commonly defined as a white blood cell (WBC) count greater than 100,000/μL, is an emergency in acute leukemia, possibly resulting in life-threatening microvascular obstruction, or leukostasis, leading to neurologic (CNS hemorrhage, thrombosis) or pulmonary (respiratory distress, hypoxia) complications. The underlying mechanisms remain poorly understood and are canonically attributed to blood hyperviscosity secondary to high WBC count and abnormal biophysical properties of leukemia cells themselves (leukemia immunophenotype, increased cell size, adhesion, and stiffness). Leukapheresis is a commonly-used therapy for rapid cytoreduction in symptomatic patients, but the procedure carries risk and existing guidelines are supported by scant evidence. Interestingly, despite hematocrit(Hct)/hemoglobin(Hgb) levels being major drivers of blood viscosity due to the high prevalence of circulating red cells (RBCs), how Hct/Hgb mediates hyperviscosity in acute leukemia is unknown. This is clinically important as Hct/Hgb often decrease as leukemic cell counts rise, and acute leukemia patients with anemia are often transfused. While sickle cell disease guidelines advise using a target post transfusion Hct of 30% to minimize iatrogenic hyperviscosity and its morbid complications, no guidelines have been established for acute leukemia. As such, can RBC transfusion actually increase leukostasis risk in acute leukemia?To explore this question requires new biophysical tools as the complexity of blood viscosity increases substantially at the microvascular level as the physical properties of the cells themselves become the major determinants of resistance to blood flow. To that end, we developed “microvasculature-on-a-chip” devices that recapitulate microvascular biophysical and hemodynamic conditions to investigate how the differing presentations of acute leukemia and transfusion support affect the effective blood viscosity at the microvascular level to cause “in vitro leukostasis.”A multiple-vessel “multiplex” microfluidic device that operates at the appropriate size scale and mimics the microvascular geometry was designed to enable assessing accurate biophysical measurements of blood hyperviscosity. The devices were microfabricated using standard polydimethylsiloxane-based photolithography (Figure 1). Acute B-cell lymphoblastic (B-ALL, 697), acute T-cell lymphoblastic (T-ALL, Jurkat) and acute myelocytic (AML, HL60) leukemia cell lines were maintained via standard cell culture conditions. Patient samples were obtained through our institution's IRB. RBCs from healthy donors were isolated and mixed with leukemia cells to achieve target Hct/Hgb and WBC levels. Various physiologic leukemia “mixtures” were then perfused under physiologic microcirculatory flow conditions through the microvascular device and microchannels occlusion was tracked via videomicroscopy (Figure 2).Using a standard least squares multivariable linear regression with first and second order effects, microchannel size, Hct/Hgb, WBC count and leukemia cell type all showed statically significant effect on in vitro leukostasis, or microchannel occlusion over time, (all p values < 0.03) (Figure 3). Overall, severe anemia appears to be protective against in vitro leukostasis and there appears to be Hct/Hgb thresholds above which in vitro leukostasis becomes more prevalent, though this is different for B-ALL versus T-ALL. This is in contrast to AML, where severe anemia does not appear to offer protection against in vitro leukostasis as occlusion was appreciated at all Hct/Hgb levels.These data suggest when determining risk for leukostasis, WBC count and leukemia immunophenotype should not be the sole determinants. Here we show Hct/Hgb levels affect microvascular blood viscosity and risk for microvascular occlusion. These results may impact decisions regarding RBC transfusions and possibly initiation of leukapheresis in asymptomatic patients. Having a model to assess risk associated with RBC transfusions and informing clinicians when a patient might become at risk for leukostasis can have a significant impact on their clinical outcome, morbidity and mortality. Ongoing studies incorporating patient lymphoid and myeloid leukemia cells are needed to support this cell line data. [Display omitted] DisclosuresKemp: Parthenon Therapeutics: Membership on an entity's Board of Directors or advisory committees. Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company.

Combining Arsenic Trioxide and Mitocans Selectively Disrupts Cellular Energetics in Acute Myeloid Leukemia

In our earlier work with arsenic trioxide (ATO) resistance in acute promyelocytic leukemia (APL), we observed that ATO resistant cells reprogrammed their metabolism from glycolysis to oxidative phosphorylation (OXPHOS) as a mechanism of resistance. We further demonstrated that it could be overcome by targeting this metabolic switch using FCCP (mitocan) in combination with ATO (Balasundaram N et al. Biorxiv 2020). There is increasing evidence that acute myeloid leukemia (AML) cells have a greater metabolic plasticity unlike ATO resistant APL cells and most cancers that rely on glycolysis. AML leukemic stem cells preferentially utilize OXPHOS for their survival (Lagadinou ED et al. Cell stem cell 2013).Mitocans like venetoclax used in combination with hypo-methylating agents are already well established in the management of AML (Pollyea D, et al. Nat Med 2021). ATO is also an effective glycolytic inhibitor (Zhang H, et al. PNAS 2015) hence we hypothesized that a combination of ATO and mitocans could potentially target the metabolic plasticity of AML cells. As the combination of ATO and FCCP was found to be non-specific we performed a small-scale screening on an AML cell line (U937) using FDA-approved compounds that are reported to target mitochondria (Gohil V et al. Nature Biotechnology, 2010). Though most of the mitocans showed predicted synergy with ATO. We focused on artesunate (ART) as a candidate for further evaluation due to its specificity for malignant cells, high therapeutic index, bioavailability, route of administration, cost-effectiveness, and global usage as an antimalarial.The combination of ATO+ART significantly reduced the viability of different subtypes of AML cell lines (THP-1, MV4:11, and Kasumi-1) and acute lymphoblastic leukemia cell lines (Jurkat E6.1, SUP B15, and MOLT-4) with minimal effect on the normal cells (CD34 and peripheral blood mononuclear cells; n=10; 48hours) (figure 1a). We noted that the selective specificity of the combination was primarily due to the iron metabolism of the leukemic cells and a requirement of iron for the activity of ART. When an iron chelator deferoxamine (DFO) was used in combination with ATO+ART there was a significant reduction in the activity of the combination on the AML cells (Figure 1b, U937; n=10; 48hours). Seahorse extracellular flux analysis validated that ART (5uM) as a single agent promoted uncoupled mitochondrial respiration and the addition of ATO resulted in a metabolic catastrophe (figure 1c and d).Chemical drug proteomic analysis using biotinylated artesunate and pull down from the leukemic cells revealed that the top interacting partners were localized in the mitochondria. We also noted that ART treatment significantly affected the mitochondrial dynamics of leukemic cells, where ART and ATO+ART treated cells had fragmented mitochondria in comparison to the control and ATO alone treated cells where the mitochondria were more elongated (figure 1e, U937 cells; n=3). We evaluated the effect of ATO+ART and their combination with azacytidine (triplet) in-vitro. Dual and triple combinations showed greater toxicity on AML cell lines and primary AML cells (Figure 1f, n=50) in comparison to the normal peripheral blood mononuclear cells (PBMNCs) and normal CD34+ cells. Taken together, these findings highlight the selective specificity of these combinations and its clinical potential in AML. [Display omitted] DisclosuresAugustin: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor. Krishna: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; KCM Vellore: Patents & Royalties; SGUL: Patents & Royalties; AML: Other: Co-Inventor. Mathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

Overexpression of Rorγt in CAR T Cells Improves Persistence and Reduces Exhaustion

Although T cells modified with a chimeric antigen receptor (CAR) are highly effective, durability of remissions may be limited by the poor persistence of adoptively transferred CAR T cells in patients. Growing evidence supports the idea that the pre-infusion phenotype of CAR T cells affects the efficacy of CAR T cell therapies, in part due to the increased ability of certain subsets of CAR T cells to persist long-term. Studies using conventional T cells have suggested that adoptive transfer of T cells programmed to have Th17/Tc17 effector functions can increase the anti-tumor efficacy and persistence of transferred cells when compared to adoptive transfer of non-Th17/Tc17 cells. However, standard CAR formats currently in clinical use that contain either a CD28 or 4-1BB costimulatory domain fail to generate substantial numbers of Th17/Tc17-like cells. Thus, few studies have investigated whether Th17/Tc17 CAR T cells could enhance the efficacy of CAR T cell therapies.We generated a multi-cistronic lentiviral construct encoding ROR-related Orphan Receptor gamma (RORγt), a key lineage-specifying transcription factor involved in Th17/Tc17 differentiation, followed by a murine CAR targeting CD19 that contains a CD28 costimulatory domain (1928z). RORγt expression did not hinder degranulation in response to CAR antigen upon co-culture with E2aPbx, a murine acute lymphoblastic leukemia cell line. Significantly higher numbers of RORγt-1928z CAR T cells produce IL-17 compared to 1928z CAR T cells (p<0.0001) suggesting that RORγt expression is sufficient to program type-17 effector functions in CAR T cells.To determine the impact of RORγt overexpression on CAR T cell phenotype and persistence in vivo, we adoptively transferred 1x10 6 1928z or RORγt-1928z CAR T cells into leukemia-bearing C57Bl6/J recipients and analyzed CAR T cells from the bone marrow at days 4, 11, and 31. As early as day 4 post-transfer, RORγt-1928z CAR T cells were present at significantly higher numbers than 1928z CAR T cells, and remained higher through Day 31 (0.16% of the bone marrow for 1928z CAR T cells vs 0.84% of the bone marrow for RORγt-1928z CARs; p= 0.0012, n=5 mice per group. The RORγt-1928z CAR T cells were enriched for an effector/effector memory phenotype (CD62L-) compared to 1928z CAR T cells, which were mostly of a central memory phenotype (CD62L+). Interestingly, the RORγt-1928z CAR T cells were enriched for IL-7Rα+ cells and expressed more IL7Rα than their 1928z CAR T cell counterparts (p=0.0012), suggesting an increased capacity for self-renewal in RORγt-1928z CAR T cells.Previous work from our lab has demonstrated that CAR T cells that persist after leukemia clearance have an exhausted phenotype, with increased expression of PD1 and a reduced functionality 1. After leukemia clearance, RORγt-1928z CAR T cells expressed lower levels of PD1 than their 1928z counterparts (p<0.0001). Finally, 1928z CAR T cells form a population of Eomes hiPD1 hi cells, associated with terminal exhaustion, that is absent in the RORγt-1928z CAR T cells.Taken together, our preliminary data demonstrates that overexpression of RORγt is sufficient to program type-17 effector functions in CAR T cells and suggests that RORγt overexpression can enhance the ability of CAR T cells to persist and self-renew, and prevent terminal exhaustion. RORγt overexpression may enhance persistence of CAR T cells and durability of remissions in hematologic malignancies. Furthermore, RORγt overexpression may also be a strategy to enhance CAR T cell efficacy against solid tumors, where an immunosuppressive microenvironment contributes to T cell dysfunction.1.Yinmeng Yang, Mark Eric Kohler, Terry J. Fry; Effect of Chronic Endogenous Antigen Stimulation on CAR T Cell Persistence and Memory Formation. Blood2017; 130 (Supplement 1): 166. doi: https://doi.org/10.1182/blood.V130.Suppl_1.166.16 DisclosuresFry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

Antibacterial and antitumoral properties of 1,2,3-triazolo fused triterpenes and their mechanism of inhibiting the proliferation of HL-60cells.

Antimicrobial resistance and cancer are two important problems affecting human health. Actively developing novel antibiotics and anticancer medicines is a priority. Natural pentacyclic triterpenoids have attracted wide attention due to their significant biological activities. In this study, a series of 1,2,3-triazolo fused triterpenoids (betulin, oleanolic acid and ursolic acid) were functionalized on the A-ring by an in-house developed multi-component triazolization reaction. The compounds were investigated for antitumoral activity in twelve cancer cell lines and were also tested for antibacterial activity against four bacteria. In terms of anticancer effects, compounds 5b-f and 8a-d displayed strong cytotoxic activity in pancreatic adenocarcinoma (Capan-1), chronic myeloid leukemia (Hap-1), acute myeloid leukemia (HL-60), acute lymphoblastic leukemia (Jurkat) and non-Hodgkin lymphoma (Rec-1) cell lines. Among them, compound 5f exhibited the most potent antiproliferative effect on HL-60cells. Further pharmacological research confirmed that compound 5f caused mitochondrial dysfunction and arrested the cell cycle in the G0/G1 phase to induce apoptosis of HL-60cells. In addition, compound 5f also induced autophagy to inhibit the proliferation of HL-60cells. Antibacterial screening revealed that compounds 2a-g and 5a-d showed modest activity against Gram-negative bacteria (Escherichia coli and Salmonella enterica subsp. enterica) with especially compounds 2c and 2d being potent inhibitors of Salmonella enterica subsp. enterica growth. Because of their promising anticancer and antibacterial activity, this series of compounds deserve further study.

Investigation the Cytotoxicity of 5-AZA on Acute Lymphoblastic Leukemia Cell Line In Vitro and Characterization the Underlying Molecular Mechanisms of Cell Death and Motility.

DNA methylation is a reversible biochemical process determinant of gene expression that is frequently observed in acute lymphoblastic leukemia (ALL). This is believed to arise from aberrant DNA methyltransferase activity establishing abnormal levels of DNA methylation in tumor cells. DNA methyltransferase inhibitor, 5-azacytidine (5-AZA), is a clinically used epigenetic drug which induces promoter demethylation and gene re-expression in human cancers. In this study, we investigated the cytotoxicity of on MOLT4 and Jurkat leukemic cell line in vitro and characterized the underlying molecular mechanisms of cell death and motility. MOLT4 and Jurkat cells were treated with 5-AZA for 12, 24 and 48 hours. The effect of the 5-AZA treatment on cell viability (MTT assay), apoptosis (annexin V/PI staining), microRNA (miRNA) and mRNA expression (real-time PCR) was measured. The results showed that 5-AZA could induce MOLT4 and Jurkat apoptotic cell death in vitro in a time-dependent manner and probably via apoptotic mechanisms. We found that treatment with 5-AZA could increase the expression of epigenetically silenced miRNAs, miR-34a, miR-34b and miR-124-1 in treated cells. In addition, mRNA analyses demonstrated that MOLT4 and jurkat cells, expressed p53 gene more than 10-fold higher compared with untreated cells in three independent experiments while the cells suppressed the expression of a subset of functionally related genes including MYC, BCL2, APEX, SIRT1, SNAIL1 and vimentin to some extent, following 5-AZA treatment. We found that a miRNAs expression level in treated cell lines was closely correlated to the expression of their target genes. Together, these findings suggest that 5-AZA may affect the viability of MOLT4 and jurkat cells, at least in part, by regulating the transcription of genes that are associated with cellular apoptotic response.

Combination therapy with miR-34a and doxorubicin synergistically induced apoptosis in T-cell acute lymphoblastic leukemia cell line.

MicroRNAs are identified to take actively part in the development of different cancers. Reduced expression of tumor suppressor miRNAs leads to cancer cell development, so restoring the expression of these miRNAs can be an appropriate treatment option for cancer. Due to the heterogeneity of cancer cells, single-drug therapy often results in drug resistance. Therefore, the combination of chemotherapy with miRNA can be a powerful strategy for cancer treatment. In the current investigation, miR-34a mimic, and negative control were purchased and transfected using jetPEI reagents. Then the synergic effects of miR-34a in combination with doxorubicin were investigated on cell death of acute T-cell lymphoblastic leukemia Jurkat cell line, as well as the expression of some genes including Caspase-3, Bcl-2, and p53 which are involved in apoptosis. Our outcomes showed that this combination remarkably reduced the expression of the Bcl-2 gene, the target gene of miR-34a. According to the results of the MTT assay, the survival rate was significantly decreased compared to the untreated cells. Results of the flow cytometry assay and DAPI staining demonstrated an increased apoptosis rate of Jurkat cells in combination therapy. Moreover, cell cycle arrest was observed at the G2/M phase in cells that were treated with miR-34a/doxorubicin. Most importantly, we showed that the transfection of the Jurkat cells with miR-34a increased the sensitivity of these cells to doxorubicin. Furthermore, the combination of miR-34a and doxorubicin drug effectively increased apoptosis of treated cells. Therefore, this method can be used as an impressive treatment for T-ALL.

Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells

BackgroundSAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 inhibitor, in the context of SAMHD1.MethodsCNDAC was tested in 13 acute myeloid leukaemia (AML) cell lines, in 26 acute lymphoblastic leukaemia (ALL) cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation.ResultsAlthough the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance.ConclusionIntrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.

The Expression Level of CCDC26 and FOXCUT Genes in Acute Lymphoblastic Leukemia

Background and Aim: Acute lymphoblastic leukemia (ALL) is a malignant disease of lymphoid progenitor cells affecting both children and adults. Long non-coding RNAs (lncRNAs) are one kind of non-coding RNAs (ncRNAs), reported modulating the initiation or progression of diverse cancers. However, the role of CCDC26 and FOXCUT long non-coding RNAs in ALL has been unknown. In this study, we explored the expression of FOXCUT and CCDC26 lncRNAs in acute lymphoblastic leukemia cell lines. Methods: Acute T lymphoblastic leukemia cell lines, RPMI 8402, Jurkat, B lymphoblastic leukemia, Daudi, and Ramos cell lines were used. After culturing the cells, RNA extraction and cDNA synthesis were performed. The real-time PCR technique was then used to study the expression of CCDC26, FOXCUT, C-kit, and FOXC1 genes. Result: We found a significant increase of CCDC26 expression in RPMI 8402 (p <0.0001) and Ramos (p <0.05) cell lines compared to the control, while decreased expression of these genes was observed in Jurkat and Daudi cell lines. Furthermore, FOXCUT gene had a significant increase in expression in all cell lines compared to the control (p <0.01 in Daudi and RPMI 8402 cell lines) (p <0.001 in Jurkat and Ramos cell lines). Conclusion: Our results demonstrated that CCDC26 and FOXCUT genes can play a regulatory role in acute lymphoblastic leukemia and may serve as a potential diagnostic biomarker and therapeutic target of acute lymphoblastic leukemia. *Corresponding Author: Seyed Hadi Mousavi, Email: hmousavi@tums.ac.ir; ORCID: https://orcid.org/0000-0002-4686-5232 Please cite this article as: Barati S, Mousavi SH, Motallebzadeh-Khanmiri J, Khani-Eshratabdi M. The Expression Level of CCDC26 and FOXCUT Genes in Acute Lymphoblastic Leukemia. Arch Med Lab Sci. 2021;7:1-9 (e16). https://doi.org/10.22037/amls.v7.33294

Establishment of Cytarabine-resistant Acute Lymphoblastic Leukemia Cell Lines and Its Resistance Mechanism

To establish cytarabine-resistant acute lymphoblastic leukemia (ALL) cell lines and investigate its possible resistant mechanism. Low-concentration cytarabine (Ara-C) continuously induced and cultured Jurkat and Nalm-6 cells to construct cytarabine-resistant cell lines Jurkat/Ara-C and Nalm-6/Ara-C. The cell viability was detected by CCK-8 assay, and the distribution of cell cycle was detected by flow cytometry. Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of multidrug resistant gene and Ara-C metabolic enzymes. The expression levels of cyclin were detected by Western blot. Jurkat/Ara-C and Nalm-6/Ara-C drug-resistant cell lines were successfully established, the resistance index of which was 1 973.908±161.163 and 7 231.643± 1 190.624, respectively. Drug-resistant cell lines had no cross-resistance to commonly used chemotherapeutic drugs, such as doxorubicin. Flow cytometry showed that the ratio of G0/G1 phase in Jurkat/Ara-C cells increased but G2/M phase decreased (P<0.05), while the cell cycle distribution of Nalm-6/Ara-C cells did not change in comparison with Nalm-6 cells. The results of real-time quantitative PCR showed that the expression of deoxycytidine kinase (DCK) and cytidine deaminase (CDA) were significantly down-regulated in drug-resistant cells (P<0.05), MRP was up-regulated in Nalm-6/Ara-C cells (P<0.05), while MDR1, LRP and BCRP did not increase in comparison with parental cells. Western blot analysis revealed that cyclinB1 was significantly under-expressed in drug-resistant cells (P<0.05), while cyclinD1 did not change, when compared with parental cells. Cytarabine-resistant ALL cell lines are successfully established by using low concentration continuous induction method, and its drug-resistant mechanism may be related to the deficiencies of DCK and cyclinB1.