Lymphocytic Leukemia Cell Line Research Articles

Analysis of Cytotoxic Granules and Constitutively Produced Extracellular Vesicles from Large Granular Lymphocytic Leukemia Cell Lines.

Large granular lymphocyte leukemias (LGLLs) are rare lymphoproliferative malignancies caused by clonal expansion of granular lymphocytes. T-cell LGLL and natural killer (NK) cell LGLL are defined based on their cellular origin. Their clinical manifestation and pathophysiology vary depending on the subtype and include, e.g., neutropenia, anemia, recurrent infections, and autoimmunity. A limited number of available patient-derived cell lines are considered valuable tools to study the biology of these malignancies. They differ in the expression of lineage-specific surface markers, but generally contain cytotoxic effector molecules in characteristic granules. We investigated the presence and release of lysosome-associated effector proteins in patient-derived LGLL cell lines by flow and imaging cytometry, by Western blotting and by bottom-up proteomics profiling. The tested cell lines did not express FasL (CD178), but did express CD26/DPP4+. Intracellularly, we detected major differences in the abundance and subcellular distribution of granzymes, perforin, and granulysin. Similar differences were seen in enriched lysosome-related effector vesicles (LREVs). The proteomics profiling of enriched EVs from an NK-LGLL line (NKL) and a T-LGLL line (MOTN-1), confirmed individual profiles of effector molecules. Our analyses underscore the individual distribution of effector proteins but also open new routes to define the role of intra- and extracellular granules in the disease manifestation or pathology of LGLLs.

Two Novel Precursors of HIV-1 Proteases Inhibitors as a Novel Anticancer Treatment on Multiple Myeloma and Chronic Lymphocytic Leukemia Cell Lines

Two Novel Precursors of HIV-1 Proteases Inhibitors as a Novel Anticancer Treatment on Multiple Myeloma and Chronic Lymphocytic Leukemia Cell Lines

Anti-Cancer Effects of CDK4/6 Inhibitor LEE011 and Chemotherapy Drugs on Lymphocytic Leukemia L1210 Cells.

Chronic lymphocytic leukemia is a slowly-progressing disease in which symptoms often do not manifest until years after disease onset. In advanced stages, infection and bleeding are common. Past studies have shown that the interaction between CDK4/6 inhibitors and chemotherapy drugs can enhance the anti-tumor efficacy of drugs and limit toxicity. Therefore, in this study, the treatment effects of combining the CDK4/6 inhibitor LEE011 with chemotherapy drugs bendamustine or hydroxyurea were investigated in vitro. The mouse lymphocytic leukemia cell line L1210 was treated with LEE011 combined with hydroxyurea or bendamustine. Western blot and flow cytometry were performed to elucidate the mechanisms behind tumor suppression. LEE011 combined with hydroxyurea or bendamustine significantly inhibited proliferation of L1210 cell lines in a concentration- and time-dependent manner as well as increased the arrest of cells in G1 and S phases. The combination of LEE011 with hydroxyurea also reduced the phosphorylation of Rb while increased the expression of total Rb protein. Furthermore, reduced expression of GPX4, which is a key protein in ferroptosis, indicates that the tumor suppression effects of this drug combination could involve ferroptosis. CDK4/6 inhibitor LEE011 treatment alone may not be a suitable treatment option for lymphocytic leukemia; however, our findings in vitro support the combination of LEE011 with chemotherapy drugs to enhance anti-tumor activity in lymphocytic leukemia.

Toll-like receptor 9 signaling in chronic lymphocytic leukemia cell lines.

Chronic lymphocytic leukemia (CLL) is a prototypic neoplasia in which malignant cells strongly depend on microenvironmental stimulations in the lymphoid tissues where they accumulate; leukemic cells are exposed to interaction with bystander and accessory cells, as well as inflammatory soluble mediators. Cell lines are frequently used to model the pathobiology of this disease; however, they do not always recapitulate leukemic cell growth and response to stimulation, and no data are available on Toll-like receptors (TLR) signaling in CLL cell lines. To address this gap, we analyzed HG3, MEC2, and PCL12 cell lines, before and after CpG stimulation, by RNA-sequencing followed by bioinformatic analyses and validation experiments. We identified NFKBIZ mRNA and the corresponding IkBz protein as robust markers of TLR9 activation in both MEC2 and PCL12, but not in HG3 cells. Next, we compared our current results with previous results obtained with primary CLL patient samples and were able to conclude that MEC2 is most similar to the patients' cells in terms of global responsiveness to TLR stimulation; in particular, MEC2 better resembles the samples of patients, as it is characterized by high expression levels of IkBz, but with a lower number of genes regulated.

The effect of HYPE knock-out on the AMPylome of human OSU-CLL leukemia cells*

In humans, AMPylation of cellular proteins is carried out by Huntingtin yeast-interacting protein E (HYPE), activated under conditions of endoplasmic reticulum stress, such as in cancer cells. Extracts of the human chronic lymphocytic leukemia cell line, OSU-CLL, were fractionated using immuno-precipitation with antibodies against adenosine-phosphate and then AMP-Tyr. The proteins isolated were modified with AMP, the ‘AMPylome.’ AMP-labelled peptides isolated from HYPE wild-type (WT) and HYPE knock-out (KO) cells were identified using tandem mass spectrometry. A total of 213 proteins were identified from WT cell extracts, while only 23 of these were pulled down from KO cells, consistent with the presence of another AMPylator, besides HYPE. The KO cells were more sensitive to fludarabine nucleoside (2-FaraA) than WT cells. Ingenuity Pathway Analysis of the AMPylated proteins identified in WT cells clustered actin binding proteins of the cytoskeleton, and proteins of the RHO GTPase pathway that would jointly stimulate cell proliferation.

Novel circular RNAs of the apoptosis-related BAX and BCL2L12 genes identified in a chronic lymphocytic leukemia cell line using nanopore sequencing.

Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key regulators of gene expression, with deregulated expression and established involvement in leukemia. The products of BCL2 and its homologs, including BAX and BCL2L12, are implicated in chronic lymphocytic leukemia (CLL). However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their role in CLL. We sought to further elucidate the contribution of BAX and BCL2L12 in CLL by unraveling the identity, localization, and potential role of their circRNAs. Therefore, total RNA from the EHEB cell line and peripheral blood mononuclear cells (PBMCs) of CLL patients and non-leukemic blood donors was extracted and reverse-transcribed using random hexamers. Next, nested PCRs with divergent primers were performed and the purified PCR products were subjected to 3rd generation nanopore sequencing. Nested PCRs were also applied to first-strand cDNAs synthesized from total RNA extracts of PBMCs from CLL patients and non-leukemic blood donors. Lastly, a single-molecule resolution fluorescent in situ hybridization (smFISH) method called circFISH was used to visualize the circRNA distribution in EHEB cells. We discovered several novel circRNAs produced by BAX and BCL2L12, which were characterized by great exon structure diversity. In addition, intriguing findings regarding their formation emerged. Interestingly, visualization of the most abundant circRNAs showed distinct intracellular localization. Moreover, a complex BAX and BCL2L12 circRNA expression pattern was revealed in CLL patients and non-leukemic blood donors. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL.

Studying the DNA damage response pathway in hematopoietic canine cancer cell lines, a necessary step for finding targets to generate new therapies to treat cancer in dogs.

Dogs present a significant opportunity for studies in comparative oncology. However, the study of cancer biology phenomena in canine cells is currently limited by restricted availability of validated antibody reagents and techniques. Here, we provide an initial characterization of the expression and activity of key components of the DNA Damage Response (DDR) in a panel of hematopoietic canine cancer cell lines, with the use of commercially available antibody reagents. The techniques used for this validation analysis were western blot, qPCR, and DNA combing assay. Substantial variations in both the basal expression (ATR, Claspin, Chk1, and Rad51) and agonist-induced activation (p-Chk1) of DDR components were observed in canine cancer cell lines. The expression was stronger in the CLBL-1 (B-cell lymphoma) and CLB70 (B-cell chronic lymphocytic leukemia) cell lines than in the GL-1 (B-cell leukemia) cell line, but the biological significance of these differences requires further investigation. We also validated methodologies for quantifying DNA replication dynamics in hematopoietic canine cancer cell lines, and found that the GL-1 cell line presented a higher replication fork speed than the CLBL-1 cell line, but that both showed a tendency to replication fork asymmetry. These findings will inform future studies on cancer biology, which will facilitate progress in developing novel anticancer therapies for canine patients. They can also provide new knowledge in human oncology.

Establishment and Characterization of an Acute Lymphocytic Leukemia Cell Line Expressing CD13 and CD33 with a Complex Philadelphia Translocation.

Objective To investigate the pathogenesis of Philadelphia (Ph)-positive acute lymphocytic leukemia (ALL), we established a lymphoblastoid cell line. Methods Bone marrow cells from a patient with Ph-positive ALL were enriched by Ficoll-Hypaque centrifugation and cultured in medium with fetal calf serum. Materials The mononuclear cells of bone marrow aspirate were obtained from an adult man with ALL after he experienced relapse following induction therapy including imatinib mesylate. Results The cell line termed TNA-M was established, carrying a three-way Ph translocation involving two chromosome 9s and one chromosome 22 as a sole karyotypic abnormality. Furthermore, the cells were positive for CD13 and CD33 in addition to CD19, CD22 and CD79a antigens. Conclusion This unique cell line is expected to be a valuable tool for understanding the pathogenesis of Ph-positive ALL.

Statins markedly potentiate aminopeptidase inhibitor activity against (drug-resistant) human acute myeloid leukemia cells.

Aim: This study aimed to decipher the molecular mechanism underlying the synergistic effect of inhibitors of the mevalonate-cholesterol pathway (i.e., statins) and aminopeptidase inhibitors (APis) on APi-sensitive and -resistant acute myeloid leukemia (AML) cells. Methods: U937 cells and their sublines with low and high levels of acquired resistance to (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester (CHR2863), an APi prodrug, served as main AML cell line models. Drug combination effects were assessed with CHR2863 and in vitro non-toxic concentrations of various statins upon cell growth inhibition, cell cycle effects, and apoptosis induction. Mechanistic studies involved analysis of Rheb prenylation required for mTOR activation. Results: A strong synergy of CHR2863 with the statins simvastatin, fluvastatin, lovastatin, and pravastatin was demonstrated in U937 cells and two CHR2863-resistant sublines. This potent synergy between simvastatin and CHR2863 was also observed with a series of other human AML cell lines (e.g., THP1, MV4-11, and KG1), but not with acute lymphocytic leukemia or multiple solid tumor cell lines. This synergistic activity was: (i) specific for APis (e.g., CHR2863 and Bestatin), rather than for other cytotoxic agents; and (ii) corroborated by enhanced induction of apoptosis and cell cycle arrest which increased the sub-G1 fraction. Consistently, statin potentiation of CHR2863 activity was abrogated by co-administration of mevalonate and/or farnesyl pyrophosphate, suggesting the involvement of protein prenylation; this was experimentally confirmed by impaired Rheb prenylation by simvastatin. Conclusion: These novel findings suggest that the combined inhibitory effect of impaired Rheb prenylation and CHR2863-dependent mTOR inhibition instigates a potent synergistic inhibition of statins and APis on human AML cells.

LP-118, a Selective Bcl-2 Inhibitor with Tuned Bcl-Xl Activity, Causes Myeloid Differentiation and Cell Death in Acute Myeloid Leukemia (AML)

Introduction Targeted therapy against bcl-2 has proven efficacious in AML, notably with bcl-2 inhibitor venetoclax. Bcl-2 is one of several proteins in the bcl-2 protein family of anti-apoptotic proteins. Primary and adaptive resistance to venetoclax-induced apoptosis is common and can be due to increased levels of other bcl-2 family proteins besides bcl-2, notably bcl-xL and mcl-1. LP-118, a next generation oral, highly potent, selective bcl-2 inhibitor with tuned bcl-xL activity, has demonstrated preliminary efficacy in venetoclax relapsed/refractory (R/R) acute lymphocytic leukemia cell lines. Methods Cell-based viability assays (MTS) were performed in primary AML cells and AML cell lines. Colony forming unit (CFU) assays were performed on primary AML cells and CD34+ stem cells from healthy donor umbilical cord blood. An in vivo study was conducted in a MOLM-13 AML mouse model (Charles River Laboratories; 8-week-old male NGC mice were injected with AML cells day 1, started treatment day 4). Immunoblot studies were performed to measure on-target activity of LP-118 and bcl-2 family proteins. Flow cytometry and cytospins were performed on primary AML cells and AML cell lines to test for maturation. Results A 24-hour (hr) continuous exposure of LP-118 against AML cell lines demonstrated growth inhibition in all except OCI-AML3 and U937 (also resistant to venetoclax), with an IC50 in the nM range of 2.2 in MOLM-13 (venetoclax IC50 9), 3.5 in MV4-11, 0.75 in HL-60 (venetoclax IC50 2), 86.4 in K562, 7.8 in KG-1 (venetoclax IC50 30), and 4.3 in Kasumi-1 (venetoclax IC50 20). A 72-hr continuous exposure of LP-118 against primary AML cells demonstrated growth inhibition with an IC50 of 12.9uM. Venetoclax R/R primary AML cells were sensitive to LP-118 between 5 and 100uM after 72-hr continuous exposure. Primary AML cells also exhibited a 63% decrease in CFUs at 10uM dose from vehicle, while CD34+ stem cells maintained colony forming capability by CFU assay. Induction of apoptosis was observed with caspase-3 and PARP cleavage on immunoblot in AML cell lines and primary AML cells. Relative baseline levels of bcl-2 family proteins by immunoblot did not correlate with AML cell line sensitivity or resistance to LP-118. Following 4- and 24-hr exposure to LP-118 in MOLM-13, MV4-11, and Kasumi-1 cell lines, BCL-2, BCL-xL, MCL-1, and BCL-W protein levels were not increased relative to vehicle by immunoblot, but BCL2A1 levels increased 3-fold. Similar results were seen following a 24-hour exposure to LP-118 in primary AML cells. Myeloid differentiation was demonstrated in primary AML cells (n=3) and AML cell lines MOLM-13 (n=2) and Kasumi-1 (n=6) by flow cytometric detection of CD11b expression after 48- and 72-hr continuous exposure to LP-118, as well as, morphologically on cytospin images. CD11b was also detected after 48-hr continuous venetoclax exposure on Kasumi-1 cells. Limiting-cell RNA-sequencing is ongoing to identify differentially expressed genes following LP-118-induced myeloid differentiation. In a MOLM-13 mouse model, single-agent LP-118 (50mg/kg PO daily) had a statistically significant median overall survival (mOS) of 24 days compared to control (20 days), but venetoclax (100mg/kg daily for 3 days then weekly) did not (mOS 21 days). There was no significant difference in OS between LP-118+azacitidine (5mg/kg IP days 1-5 of 21-day cycle) and venetoclax+azacitidine. After 60 days, the median OS of LP-118+gilteritinib (15.24 mg/kg PO daily) was not reached and showed increased OS compared to azactidine+gilteritinib and venetoclax+azacitidine. By splenic histopathology, blasts are notably reduced in mice treated with azacitidine+gilteritnib, but spleens are effaced by neoplastic blasts in all other treatment groups. Conclusions LP-118 shows effective cell killing in multiple AML cell lines and primary samples with various molecular and cytogenetic profiles, including a patient sample with a PTPN11 mutation, against which venetoclax is known to be ineffective. Compared to venetoclax, LP-118 is effective at lower IC50 values in AML cell lines and is active in venetoclax R/R primary AML samples. LP-118 also proves efficacious in vivo, exhibiting improved OS and decreased disease burden. Finally, LP-118 induces myeloid differentiation, to our knowledge a novel mechanism of action of bcl-2/bcl-xL inhibition in AML. These data support the recently initiated AML clinical trial (NCT04771572).

Effects of Glutaminase Inhibition on Venetoclax, Ibrutinib, and AZD-5991 Resistance in Chronic Lymphocytic Leukemia Cell Lines

Introduction: Bruton tyrosine kinase and BCL-2 inhibitors are widely used in CLL, but resistance problem is highly relevant. One of the resistance mechanisms may be metabolic rewiring followed by activation of oxidative phosphorylation. Glutamine plays a major role in cancer metabolism, and glutamine limitation rather than glucose contributes to lower oxygen uptake and mediates apoptosis of cancer cells. It was shown that glutamine metabolism changes contribute to venetoclax (Chen Z. et al, Blood, 2021; Guièze R. et al. Cancer Cell, 2019) and ibrutinib (Galicia-Vázquez G. et al. Frontiers in Oncology, 2018) resistance. We investigated in vitro effects of glutaminase-1 (GLS-1) enzyme inhibition and its potential synergistic effects with venetoclax, ibrutinib, and MCL-1 inhibitor AZD-5991 on resistant cell lines. CB-839 (telaglenastat) is a potent, reversible, and selective GLS-1 inhibitor with 24 nM IC50 for recombinant human mitochondrial glutaminase C. The compound was tested in the clinic for hematological malignancies and solid tumors. Methods: We have tested CB-839 alone or in combination with venetoclax, ibrutinib, and AZD-5991 on resistant HG-3 and MEC-1 CLL cell lines. Cells were incubated with 0.01 to 100 μM of CB-839 for 72 hours. The inhibition of glutamine, glutamate, and glutathione induced by CB-839 alone was measured using a luminescence assay. DCFDA/ H2DCFDA and MitoSOX Red stainings were utilized to evaluate cellular reactive oxygen species (Total ROS) and mitochondrial superoxide (MitoSOX) accumulation, respectively. Intracellular nucleotides including ATP level were measured using high-pressure liquid chromatography. For drug combinations, MTS assay and Annexin V/Propidium Iodide staining were used to assess cell proliferation and apoptosis, respectively. The combination index (CI) for synergy was calculated using Compusyn software. Results: HG3 and MEC1 cell lines were resistant to venetoclax, ibrutinib, and AZD-5991 single agents. The IC50 values for HG3 are 8.1 μM, 1.3 μM, 9.9 μM, and for MEC1 are 2.9 μM, 1.2 μM, and 1.9 μM for venetoclax, ibrutinib, and AZD-5991, respectively. In these cell lines, CB-839 caused a dose-dependent decrease in GLS-1 activity, measured as intracellular glutamate concentration, and inhibited glutathione synthesis (Figure 1A). CB-839 treated cells showed increased MitoSOX, but not Total ROS accumulation. However, as the endpoint effect, the decrease of cell proliferation rather than apoptosis was detected. CB-839 treatment caused a mild decrease in ATP pool in both cell lines. According to MTS assay, venetoclax demonstrated only the particular synergism with CB-839 (CI <1.0 at all effective doses (ED50, ED75, ED90, ED95) for MEC-1, and at ED50, ED75 for HG-3 cell line). In addition, CB-839 increased pro-apoptotic effect of venetoclax in both cell lines. Notably, that CB-839 did not improve any ibrutinib-mediated effects on cell proliferation and apoptosis in CLL cell lines. The most distinct synergism was observed in CB-839 and AZD-5991 combination group (CI <1.0 at all ED for MEC-1 and HG-3 cell lines) with 5- and 19.8-fold shifts in IC50 (Figure 1B). To extend these results, we are performing these combinations in primary CLL cells that are sensitive or resistant to targeted agents such as ibrutinib or venetoclax. Conclusions: Combinations of CB-839 with other agents targeting multiple mechanisms have the potential to overcome metabolic adaptation. Our findings suggest that CB-839 has limited efficacy in overcoming ibrutinib CLL resistance. However, the synergism with apoptosis inducers such as venetoclax and AZD-5991 require further investigations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ganoderma lucidum polysaccharide inhibits the proliferation of leukemic cells through apoptosis.

To investigate the cytotoxic effect of polysaccharides derived from Ganoderma lucidum on T lymphocyte leukemia cells. Water-soluble polysaccharides were extracted from the fruit bodies of G. lucidum, purified, and characterized using HPGPC-MALLS and NMR. The cytotoxicity of G. lucidum polysaccharide fraction 5 (GLP5) to T lymphocyte leukemia cell line Jurkatand human immortalized epidermal cell line HaCat was assessed using MTT assay. Apoptosis was assessed using flow cytometry. Expressions of apoptosis-related genes in the cells after being exposed to GLP5 were detected using Western blot assay. GLP5 was a β-(1→3) and β-(1→6) linked glucan. It inhibited the proliferation of Jurkat cells in a concentration-dependent manner and the half-maximal inhibitory concentration (IC50) was 34.5 mg/L but did not suppress the growth of HaCat cells. Apoptotic cells in Jurkatcells were detected to increase with increasing GLP5 concentrations. The expression levels of cleaved caspase-3 were significantly higher after the cells were exposed to 25 and 50 mg/L GLP5 when compared to non-exposed cells (Control). In addition, the expression levels of BAX and Bcl2 were significantly up- and down-regulated after treatment with GLP5 at 25 and 50 mg/L when compared with control (P<0.05), respectively. GLP5 has antiproliferative activity against Jurkat cells and the activity is likely mediated through the activation of apoptosis pathways.

ASP-Enzymosomes with Saccharomyces cerevisiae Asparaginase II Expressed in Pichia pastoris: Formulation Design and In Vitro Studies of a Potential Antileukemic Drug

The bacterial enzyme asparaginase is the main treatment option for acute lymphoblastic leukemia. However, it causes side effects, such as immunological reactions, and presents undesirable glutaminase activity. As an alternative, we have been studying asparaginase II from Saccharomyces cerevisiae, coded by ASP3 gene, which was cloned and expressed in Pichia pastoris. The recombinant asparaginase (ASP) presented antileukemic activity and a glutaminase activity 100 times lower in comparison to its asparaginase activity. In this work, we describe the development of a delivery system for ASP via its covalent attachment to functionalized polyethylene glycol (PEG) polymer chains in the outer surface of liposomes (ASP-enzymosomes). This new delivery system demonstrated antiproliferative activity against K562 (chronic myeloid leukemia) and Jurkat (acute lymphocytic leukemia) cell lines similar to that of ASP. The antiproliferative response of the ASP-enzymosomes against the Jurkat cells suggests equivalence to that of the free Escherichia coli commercial asparaginase (Aginasa®). Moreover, the ASP-enzymosomes were stable at 4 °C with no significant loss of activity within 4 days and retained 82% activity up to 37 days. Therefore, ASP-enzymosomes are a promising antileukemic drug.

STL1, a New AKT Inhibitor, Synergizes with Flavonoid Quercetin in Enhancing Cell Death in A Chronic Lymphocytic Leukemia Cell Line.

Using a pharmacophore model based on the experimental structure of AKT-1, we recently identified the compound STL1 (ZINC2429155) as an allosteric inhibitor of AKT-1. STL1, was able to reduce Ser473 phosphorylation, thus inhibiting the PI3K/AKT pathway. Moreover, we demonstrated that the flavonoid quercetin downregulated the phosphorylated and active form of AKT. However, in this case, quercetin inhibited the PI3K/AKT pathway by directly binding the kinases CK2 and PI3K. In the present work, we investigated the antiproliferative effects of the co-treatment quercetin plus STL1 in HG-3 cells, derived from a patient affected by chronic lymphocytic leukemia. Quercetin and STL1 in the mono-treatment maintained the capacity to inhibit AKT phosphorylation on Ser473, but did not significantly reduce cell viability. On the contrary, they activated a protective form of autophagy. When the HG-3 cells were co-treated with quercetin and STL1, their association synergistically (combination index < 1) inhibited cell growth and induced apoptosis. The combined treatment caused the switch from protective to non-protective autophagy. This work demonstrated that cytotoxicity could be enhanced in a drug-resistant cell line by combining the effects of different inhibitors acting in concert on PI3K and AKT kinases.

Immunomolecular evaluation of dihydroartemisinin effects on apoptosis in chronic lymphocytic leukemia cell lines

IntroductionDihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has recently shown to induce apoptosis in many types of cancer cells. In this study, we aimed to determine the effects of DHA on apoptosis in human chronic lymphocytic leukemia (CLL) cell lines. MethodsThe cells were treated separately and combined by DHA and Fludurabine (FLU) during 24, 48 and 72 hours. The cell viabilities determined by XTT method. Following separate and combined treatment of IC50 concentrations of DHA and FLU to the cells during 24 hours, the cells were analyzed by flow cytometry to determine the effects on apopotis staining with AnnexinV FITC and PI. mRNA and protein expression levels of TCTP, Mcl-1, Bcl-2, Bax and Caspase-3 were analyzed to find out the molecular mechanisms of apoptosis by using quantitative real-time PCR and flow cytometric methods. ResultsTreatment with DHA alone or in combination with FLU induced apoptosis in a dose dependent manner in CLL cells. DHA alone was more effective than FLU alone or combined treatment with DHA and FLU. Our results suggest that Bcl-2 protein family member Bax was active in the apoptotic response of CLL cells after DHA treatment. Moreover, the apoptotic response induced by DHA was independent from the p53 mutation status of the CLL cells. ConclusionDHA might be a potential anti-cancer therapeutic for CLL.

Optimizing extracellular vesicles' isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant.

In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture–conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/μg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.

Multifunctional barcoding with ClonMapper enables high-resolution study of clonal dynamics during tumor evolution and treatment.

Lineage-tracing methods have enabled characterization of clonal dynamics in complex populations, but generally lack the ability to integrate genomic, epigenomic and transcriptomic measurements with live-cell manipulation of specific clones of interest. We developed a functionalized lineage-tracing system, ClonMapper, which integrates DNA barcoding with single-cell RNA sequencing and clonal isolation to comprehensively characterize thousands of clones within heterogeneous populations. Using ClonMapper, we identified subpopulations of a chronic lymphocytic leukemia cell line with distinct clonal compositions, transcriptional signatures and chemotherapy survivorship trajectories; patterns that were also observed in primary human chronic lymphocytic leukemia. The ability to retrieve specific clones before, during and after treatment enabled direct measurements of clonal diversification and durable subpopulation transcriptional signatures. ClonMapper is a powerful multifunctional approach to dissect the complex clonal dynamics of tumor progression and therapeutic response.

Fbxw11 Triggers a Dual Signaling Axis to Exert Proliferation Effects on Lymphocytic Leukemia

Dysregulation of signal conduction will break the balance of normal hematopoiesis and cause various blood diseases. Ubiquitin-proteasome system (UPS) takes part in both physiological and pathological processes by post-translational regulating intracellular signal transduction pathways. Fbxw11 as a constituent of the SCF (Skp1-Cul1-F box) E3 ubiquitin ligase complex, regulates various signal transduction pathways and its pathological roles in tumorigenesis have been suggested. However the role of Fbxw11 in the development of leukemia and the underlying mechanisms remain largely unknown.To assess the clinical relevance of Fbxw11, the expression of Fbxw11 in AL patients was also studied by real-time PCR. The expression of Fbxw11 was higher in newly diagnosed ALL (p=0.0304) and relapsed ALL patients (p=0.0389) than that in healthy donors. However, results from a follow-up study demonstrated that the expression of Fbxw11 was dramatically decreased after chemotherapy achieving CR (p=0.0202). These results were conformed in the BloodSpot database.To study in vitro effects of Fbxw11 on leukemia cells, the lymphocytic leukemia cell line, L1210, was stably transfected with Fbxw11 transcript variants and defined as L1210-control, L1210-Fbxw11a, L1210-Fbxw11c and L1210-Fbxw11d, respectively. Increased percentage of S/G2/M phase cells was detected in L1210 cells with high level Fbxw11 by PI stainingand Ki-67/hoechst33342 staining. Fbxw11 significantly promoted the growth of L1210 cells. The proliferation effects of Fbxw11 on the leukemia cells were also tested in vivo . Tumor growth was significantly higher in groups with high level Fbxw11 variants. Significantly higher rate of Brdu positive cells were detected by confocal microscopy in slides formed by L1210 cells expressing high level Fbxw11 variants than control slides .To further investigate the pro-proliferation mechanism by Fbxw11 transcript variants, Agilent Sureprint G3 Mouse Gene Expression Microarray was performed after total RNA was isolated from control L1210 cells and those expressing high level Fbxw11 transcript variants. Categories of NF-κB and Wnt/β-catenin signaling pathway has high enrichment score in KEGG and IPA respectively. Categories of regulation of cell proliferation and positive regulation of T cell proliferation have high enrichment score in GO biological process. Cell cycle regulator, CyclinD1 and CyclinD3, which were regulated by multiple transcription factors including NF-κB and β-catenin, were significantly up-regulated in L1210 cells expressing high level Fbxw11 transcript variants at protein level. The expression of other NF-κB-regulated genes and β-catenin/TCF-regulated genes were also up-regulated in L1210 cells with high level Fbxw11 transcript variants, which was consistent with the array data. Activation of both NF-κB and β-catenin/TCF signaling pathways was detected in 293T cells expressing high level Fbxw11 transcript variants by dual luciferase reporter system. Furthermore, combination of low dose inhibitors for NF-κB and β-catenin/TCF pathways (ICG-001 and JSH-23) completely diminished pro-proliferation effects of Fbxw11 though the use of low dose either inhibitor had little effect.Taken together, Fbxw11 was up-regulated in ALL patients. High level expression of Fbxw11 stimulated the proliferation of L1210 lymphocytic leukemia cells in vitro and promoted tumor formation in vivo through cell cycle regulation. Most importantly, activation of both NF-κB and β-catenin/TCF signaling pathways were involved in this process. This work highlights dual signaling activation and illustrates the mechanism of Fbxw11 on the development of lymphocytic leukemia and implys possible approaches for leukemia therapy.The work was supported by the Grants 81300376, 81370634, 81570153, from the National Natural Science Foundation of China (NSFC); 14JCQNJC10600 and 17 JCZDJC35000 from the Tianjin Science and Technology Programs DisclosuresNo relevant conflicts of interest to declare.

Cytarabine and EIP co-administration synergistically reduces viability of acute lymphoblastic leukemia cells with high ERG expression

The E26 transformation sequence-related gene ERG encodes a transcription factor involved in normal hematopoiesis, and its expression is abnormal in leukemia. Especially in a type of acute lymphoblastic leukemia (ALL) that is refractory and easy to relapse, the expression of ERG protein is abnormally increased. Chemotherapy can alleviate the condition of ALL, but the location and survival mechanism of the remaining ALL cells after chemotherapy are still not fully understood. It is becoming increasingly clear that the interaction between leukemia cells and their microenvironment plays an important role in the acquisition of drug resistance mutations and disease recurrence. We selected an acute lymphocytic leukemia cell line with high ERG expression, and studied the synergistic effect of chemotherapeutics and small molecule peptides through cell proliferation, apoptosis, and cell cycle experiments; At the same time, we inoculated acute lymphocytic leukemia cells with high ERG expression into mice with severe immunodeficiency to simulate human ALL and investigated (i) the effects of co-administration on the nesting and invasion of leukemia cells and (ii) the effects of the small molecule peptide drug EIP, which targets ERG, on the sensitivity of ALL chemotherapy and the underlying mechanisms.Ara-c and EIP synergistically reduces viability of ALL cells with high ERG expression may be achieved by promoting their apoptosis and inhibiting their nesting.

Antitumor effects of sodium selenite on acute lymphocytic leukemia.

Selenium is obligatory for proper functioning of body as it is the part of enzyme protection system. Its both organic and inorganic forms are thought to be active as an antitumor agent. We trialed the different dosages (0 × 106 M, 2.7 × 106 M, 5.4 × 106 M, and 8.1 × 106 M) of sodium selenite given to the acute lymphocytic leukemia cell lines incubated for 24, 48, and 72 h. The ratios of dead cells to live cells when treated with sodium selenite were very high as compared to the control with no treatment. This dosage-dependent apoptosis increased with the incubation time.