Human Acute Lymphoblastic Leukemia Cells Research Articles

Abstract A81: Early TLR-mediated killing of leukemia in bone marrow is correlated with durable protection against B cell precursor acute lymphoblastic leukemia

Abstract The ability of Toll-like receptor (TLR)-induced innate immune responses to activate adaptive immune responses offers considerable therapeutic potential for cancer treatment, most likely as part of combination therapies. The potent immunostimulatory capacity of TLR agonists provides means to generate antigen-specific antitumor T cell responses from antigen-nonspecific innate immune stimulation. B cell precursor (BCP) acute lymphoblastic leukemia (ALL) exhibits the very low mutational burden common to other pediatric cancers. Despite this limited neo-antigenicity, we previously reported the ability of CpG oligodioxynucleotides (ODN), a ligand for TLR9, to mediate anti-leukemia activities that establish long-term protection against ALL outgrowth in a transplantable syngeneic model using cell lines. CpG ODN also reduced the burden of primary human ALL in mouse xenografts, primarily via induction of NK cell-mediated cytotoxicity. However, our recent demonstration that endosomal TLR agonists generated distinct effects on pre-leukemic BCP cells, coupled with the divergent effects of TLR ligands on the immunogenicity of primary human ALL cells suggest that there are significant variables that contribute to the overall outcome of TLR stimulation in the context of BCP cell malignancy. In this study, we compared the ligands for TLR3 (polyI:C), TLR7/8(R848) and TLR9 (CpG ODN) for their ability to elicit control of primary ALL cells and correlated protection with early innate immune activation to identify the necessary component of a long-term protective anti-leukemic response. We adoptively transferred syngeneic primary ALL cells to wild-type mice then administered TLR ligands starting at day 7 for 3 doses every 4 days. 21 days after leukemia challenge, we evaluated the disease burden in the peripheral blood, spleen, and bone marrow of recipients. While a significantly lower disease burden was observed in all three anatomical compartments of mice treated with polyI:C, CpG ODN- and R848-treated mice failed to control the early outgrowth of transferred cells in the spleen and bone marrow, respectively. While R848-treated mice had a slightly delayed disease progression with median survival (MS) of 40 days compared to that of 28 days of PBS-treated control group, CpG ODN treatment conferred a significant survival advantage where over 57% of treated mice achieved a prolonged disease-free status. Despite successfully inducing systemic anti-ALL responses, poly I:C treatment failed to confer durable protection, achieving only a modest increase in disease-free survival in treated mice (MS=42 days). Moreover, CpG ODN-induced long-term survival in the absence of effective early immune-mediated control of ALL in spleen implies that bone marrow is not only an important homing niche supporting the survival and proliferation of leukemia blasts prior to migration into the circulation, but a critical target site for controlling disease progression. Furthermore, to investigate the early innate immune responses associated with the differential overall effects induced by TLR agonists, we evaluated the activation status of innate immune system using RAG-knockout mice. A single administration of CpG ODN, but not polyI:C nor R848, was sufficient to rapidly induce anti-ALL immune responses involving activated natural killer and antigen-presenting cells in the bone marrow of the mice bearing ALL in 4 days. This suggests that CpG ODN has superior capacity to initiate innate immune-mediated anti-ALL immune responses and activate subsequent antigen-specific T cell responses. TLR-mediate innate immune responses induced early in life are distinct from that induced in adult life. Given the peak incidence of pediatric ALL between 3-5 years of age, we are currently examining the influence of age on the TLR-induced anti-ALL immune responses to further evaluate the therapeutic benefits of TLR ligands. Our results demonstrate the differential magnitude of tissue-specific innate immune-mediated control of leukemia outgrowth induced by TLR stimulations correlating with durable disease-free survival. These findings provide mechanistic explanation for TLR-mediated protective immune responses and highlight the potential target site necessary for effective elimination of leukemia cells. Citation Format: Sumin Jo, Peter van den Elzen, Gregor S.D Reid. Early TLR-mediated killing of leukemia in bone marrow is correlated with durable protection against B cell precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A81.

Wee1 kinase inhibitor MK-1775 induces apoptosis of acute lymphoblastic leukemia cells and enhances the efficacy of doxorubicin involving downregulation of Notch pathway.

Acute lymphoblastic leukemia (ALL) is an aggressive hematologic malignancy affecting pediatric and adult populations. Although the outcomes of ALL in children have improved markedly in previous years, limited treatment strategies are available at present for adult patients with ALL. Wee1 is a crucial cell cycle checkpoint kinase of G2/M that regulates cell cycle progression and maintains chromatin integrity. MK-1775, a selective inhibitor of Wee1 has recently been identified to be able to induce apoptosis of tumor cells by abrogating G2/M checkpoint. The present study investigated the anti-leukemic activity of MK-1775 alone and in combination with doxorubicin (Adriamycin®; ADM) in various human ALL cell lines. MK-1775 treatment induced apoptosis of ALL cells, accompanied by unscheduled mitotic entry and downregulation of Notch pathway. The anti-leukemic activity of MK-1775 was in a concentration- and time-dependent manner. The data also indicated that it decreased the half-maximal inhibitory concentration (IC50) of ADM compared with the control group. The combination of MK-1775 and ADM induced an increased apoptotic rate compared with each agent alone. In addition, the human bone marrow stromal cell HS-5 cell line was detected to exhibit an increased IC50 value of MK-1775 treatment in contrast to ALL cell lines. It indicates that the hematopoietic supportive capability may remain intact during the treatment of MK-1775. Taken together, the Wee1 inhibitor MK-1775 may be an attractive agent in the treatment of patients with ALL.

In vitro combinatorial anti-proliferative and immunosuppressive effects of Brucea javanica extract with CX-4945 and imatinib in human T-cell acute lymphoblastic leukemia cells

Since 1970, the isolated and identified components of Brucea javanica (L.) Merr. have been known to contain anticancer effects, particularly antileukemic effect. In this study, the inhibitory effect of Brucea javanica (BJ) on cell growth and inflammation was confirmed in human T-cell acute lymphocytic leukemia (T-ALL) cells, and its efficacy as an antileukemic agent was verified. Our results showed that BJ extract induced caspase-dependent apoptosis of T-ALL Jurkat cells through inhibition of the CK2-mediated signaling pathway, while exerting no significant cytotoxicity in normal peripheral blood mononuclear cells. Moreover, BJ extract suppressed the NF-κB signaling pathway, thus, inhibiting the interleukin (IL)-2 expression induced by phorbol 12-myristate 13-acetate (PMA) and phytohemagglutinin (PHA). Notably, combined treatment with BJ extract plus CX-4945 or imatinib exerted enhanced inhibitory effects on T-ALL cell growth and IL-2 production. Overall, these results suggest that BJ extract can be a potent therapeutic herbal agent for T-ALL treatment and prevention of IL-2 mediated inflammatory immune responses.

Abstract 1783: UMG1, a novel humanized monoclonal antibody against a glysosylated-CD43-related epitope, induces antibody-dependent cellular cytotoxicity (ADCC) on human T-cell acute lymphoblastic leukemia cells

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic malignancy accounting for about 25% of all acute leukemias. Due to the poor therapeutic scenario and severe prognosis of T-ALL, novel drugs are eagerly awaited. The targeting of tumor-associated antigens by monoclonal antibodies (mAb) for induction of immune-mediated cellular cytotoxicity is presently a promising immunotherapeutic strategy. We previously developed a novel mAb direct against a heavy glycosylated oncofetal epitope of CD43 (UN1) with known potential application as therapeutic and diagnostic tool. By screening different cancer cell lines, we observed that UN1 is highly and selectively expressed on malignant T-ALL cells. The expression of UN1 was then evaluated in 38 T-ALL patient-derived blasts and high correlation for a specific subset of patients (about 80%) belonging to the cortical T-ALL group (EGIL T3) was detected. Accordingly, we developed a humanized mAb, (UMG1) and an afucosylated enginereed version (a-UMG1). Therefore, to elucidate the mechanism of action of these mAbs, we investigated complement-mediated cytotoxicity (CDC), ADCC and antibody-dependent cellular phagocytosis (ADCP). To this aim, T-ALL cells (HPB-ALL and CCRF-CEM) have been cultured in the presence of complement, peripheral blood mononuclear cells (PBMCs), NK-92-CD16+ cells or macrophages, using increasing concentrations of both mAbs. Notably, we observed that mAbs treatment alone did not exert either cellular cytotoxicity or CDC on target cells. Conversely, both mAbs induced significant ADCC mediated by PBMCs and NK-92-CD16+ cell line in term of NK degranulation and cytotoxicity and macrophage-mediated ADCP. In vivo results demonstrated a powerful activity of UMG1 in 3 different models of T-ALL in NSG mice in the presence or absence of NK-92-CD16+ cells. Specifically, in two subcutaneous models, we observed a strong ability of both mAbs to delay tumor growth and increase survival of treated mice. As expected, the combinatory treatment with NK-92-CD16+ cell line strongly improved the activity of a-UMG1. Importantly, in the orthotopic disseminated model, which better reproduces the human T-ALL disease, we observed 5 out of 20 treated mice alive and free of disease 100 days after injection. Furthermore, to explore the possibility of combination therapy, the modulation of UN1 expression by chemotherapeutic agents in T-ALL was investigated. Interestingly, we observed that methotrexate and doxorubicin , alone or in combination, increased UN1 expression at low nanomolar concentrations, and improved ADCC in vitro. In conclusion, we demonstrated that UMG1 and a-UMG1 represent a novel promising immune-therapeutic tool for the treatment of T-ALL patients. Citation Format: Cirino Botta, Maria E. Gallo Cantafio, Chiara Buracchi, Maria A. Siciliano, Maria Cucè, Caterina Riillo, Franca M. Tuccillo, Daniele Caracciolo, Emanuela Altomare, Mariamena Arbitrio, Maria T. Di Martino, Marco Rossi, Andrea Biondi, Giuseppe Gaipa, Pierosandro Tagliaferri, Pierfrancesco Tassone. UMG1, a novel humanized monoclonal antibody against a glysosylated-CD43-related epitope, induces antibody-dependent cellular cytotoxicity (ADCC) on human T-cell acute lymphoblastic leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1783.

Ultrasound-based cell sorting with microbubbles: A feasibility study.

The isolation and sorting of cells is an important process in research and hospital labs. Most large research and commercial labs incorporate fluorescently or magnetically labeled antibodies adherent to cell surface antigens for cell identification and separation. In this paper, a process is described that merges biochemical labeling with ultrasound-based separation. Instead of lasers and fluorophore tags, or magnets and magnetic particle tags, the technique uses ultrasound and microbubble tags. Streptavidin-labeled microbubbles were mixed with a human acute lymphoblastic leukemia cell line, CCL 119, conjugated with biotinylated anti-CD7 antibodies. Tagged cells were forced under ultrasound, and their displacement and velocity quantified. Differential displacement in a flow stream was quantified against erythrocytes, which showed almost no displacement under ultrasound. A model for the acoustic radiation force on the conjugated pairs compares favorably with observations. This technology may improve on current time-consuming and costly purification procedures.

Cytotoxic activity of stigmasteryl esters and products of their thermo-oxidative degradation against drug sensitive and drug resistant human acute lymphoblastic leukemia cells.

Phytosterols are mainly known as a cholesterol-lowering factor, although they form oxidation products during food storage and processing. Moreover, phytosterol oxidation products (POP) can be ab- sorbed and found in human serum, so there is the need to investigate their impact on different kinds of cells. Esters of fatty acids (oleic, linoleic and linolenic) with stigmasterol were synthetized and heated at 180°C, for 1–12 hours. The cytotoxic effect on the leukemic cells of unheated stigmasteryl esters and the mixture of compounds after heating was determined using MTT assays. POP were identified using GC-MS. The total number of POP was analysed by SPE fractionation and GC-FID separation. Dimers, trimers and oligomers in non-polar fraction were determined by gel permeation chromatography with refractive index detection. After heating, stigmasterol oxidation products were formed (up to 1.1 mg/g ester). The heating increased the potency of the compounds to reduce cell population and form POPs and oligomers in a time-dependent manner. The cytotoxicity depends on the kind of ester, dose and time. The strongest cytotoxic effect was found after 72 hours of cell treatment. Among the three stigmasteryl esters tested the most cytotoxic effect was caused by stigmasteryl linoleate.

Cytotoxic activity of stigmasteryl esters and products of their thermo-oxidative degradation against drug sensitive and drug resistant human acute lymphoblastic leukemia cells [pdf

Cytotoxic activity of stigmasteryl esters and products of their thermo-oxidative degradation against drug sensitive and drug resistant human acute lymphoblastic leukemia cells [pdf

Leukemia reconstitution in vivo is driven by cells in early cell cycle and low metabolic state

In contrast to well-established hierarchical concepts of tumor stem cells, leukemia-initiating cells in B-cell precursor acute lymphoblastic leukemia have not yet been phenotypically identified. Different subpopulations, as defined by surface markers, have shown equal abilities to reconstitute leukemia upon transplantation into immunodeficient mice. Using a non-obese diabetes/severe combined immunodeficiency human acute lymphoblastic leukemia mouse model and cell cycle analysis annotating cells to distinct cycle phases, we functionally characterized leukemia-initiating cells and found that cells in all stages of the cell cycle are able to reconstitute leukemia in vivo, with early cycling cells (G1blow population) exhibiting the highest leukemia-initiating potential. Interestingly, cells of the G2/M compartment, i.e. dividing cells, were less effective in leukemia reconstitution. Moreover, G1blow cells were more resistant to spontaneous or drug-induced cell death in vitro, were enriched for stem cell signatures and were less metabolically active, as determined by lower levels of reactive oxygen species, compared to G2/M stage cells. Our data provide new information on the biological properties of leukemia-initiating cells in B-cell precursor acute lymphoblastic leukemia and underline the concept of a stochastic model of leukemogenesis.

Down-regulation of JARID1B expression inhibits cell proliferation, induces apoptosis and blocks cell cycle in human acute lymphoblastic leukemia cells.

Acute lymphocytic leukemia (ALL) is a multi-factorial blood disease with unknown pathogenesis. Histone H3K4 methylation was significantly reduced in ALL patients, whereas jumonji AT-rich interactive domain 1B (JARID1B) was the specific demethylase of H3K4me. This study explores the expression level of JARID1B in ALL patients and down-regulated JARID1B expression in ALL cells to explore the function of JARID1B in ALL. JARID1B mRNA expression level in ALL patients was detected by Real-time PCR. The peripheral blood mononuclear cells from healthy volunteers were selected as control. JARID1B shRNA was transfected with MOLT-4 cells and BALL-1 cells. JARID1B protein expression and H3K4me2 and H3K4me3 levels were detected by Western blot assay. Cell proliferation was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Cell apoptosis and cell cycle were determined by flow cytometry. Bcl-2, Bax, Procaspase 3, and cyclin P21 expressions were evaluated by Western blot assay. JARID1B mRNA expression in primary bone marrow cells from ALL patients was significantly higher than that of healthy volunteers (p<0.05). The levels of histone H3K4me3 and H3K4me2 were up-regulated after JARID1B shRNA transfection. JARID1B shRNA significantly inhibited the proliferation of MOLT-4 and BALL-1 cells, induced apoptosis, and blocked cell cycle in G0/G1 phase compared with the control group (p<0.05). JARID1B is highly expressed in ALL. Down-regulating its expression inhibited leukemia cell proliferation, promoted apoptosis, and blocked cell cycle in G0/G1 phase through histone H3K4 methylation. JARID1B is an oncogene in ALL.

Comparison of bioactive compounds content in leaf extracts of Passiflora incarnata, P. caerulea and P. alata and in vitro cytotoxic potential on leukemia cell lines

Passiflora caerulea L., P. alata Curtis and P. incarnata L. (synonym for P. edulis Sims), are the most popular representatives of the Passiflora genus in South America. In recent years, a growing attention is paid to the biological activity and phytochemical profiles of crude extracts from various species of Passiflora in worldwide. The aim of this study was to evaluate and to compare of anti-leukemic activity of the dry crude extracts from leaves of three Passiflora species from greenhouse of Poland in two human acute lymphoblastic leukemia cell lines: CCRF-CEM and its multidrug resistant variant. Two systems of liquid chromatography in order to assessment of phytochemical composition of extracts were applied. Extracts of P. alata and P. incarnata showed the potent inhibitory activity against human acute lymphoblastic leukemia CCRF-CEM, while P. caerulea not showed activity (or activity was poor). Despite similarities in quality phytochemical profile of extracts from P. caerulea and P. incarnata, differences in quantity of chemical compounds may determine their various pharmacological potency. For the activity of P. alata extract the highest content of terpenoids and a lack of flavones C-glycosides are believed to be crucial. Summarizing, the crude extract from P. alata leaves may be considered as a substance for complementary therapy for cancer patients.

Effect of 4' -Hydroxywogonin on Proliferation and Apoptosis of Human Acute Lymphoblastic Leukemia Cells

To investigate the effect of 4' -hydroxywogonin on proliferation and apoptosis of human acute lymphoblastic leukemia SUP-B15 and Jurkat cells, and to analyze its possible mechanism. SUP-B15 and Jurkat cells were cultivated in vitro and treated with different concentrations of 4' -hydroxywogonin, the inhibitory effect of 4' -hydroxywogonin on the proliferation of SUP-B15 and Jurkat cells was detected by CCK-8 method; the cell apoptosis was examined by the flow cytometry with Annexin V-APC/7-AAD donble staining; the expression of C-MYC, BCL-2 and cleaved caspase 3 in SUP-B15 and Jurkat cells were measured with Western blot. 4' -hydroxywogonin inhibited the proliferation of SUP-B15 and Jurkat cells in a dose-dependent manner (r=0.78, r=0.89), with IC50 value of (6.32± 0.53) µg/ml in SUP-B15 cells and (12.04± 0.42) µg/ml in Jurkat cells at 24 h. The early apoptotic rate of cell was also enhanced with the increase of 4' -hydroxywogonin concentrations. The results of Western blot showed that 4' -hydroxywogonin could down-regulate the expression of proliferation-related molecule C-MYC(P<0.01) and apoptosis-related molecule BCL-2(P<0.01), the expression of apoptosis-related molecule cleaved caspase 3 was up-regulate(P<0.01). 4' -hydroxywogonin shows the effects of anti-tumor by inducing cell apoptosis and inhibiting cell proliferation, its molecular mechanism maybe relate with down-regulation of C-MYC and BCL-2 expression and up-regulation of the cleaved caspase 3 expression.

Effect of Anacardium occidentale leaf extract on human acute lymphoblastic leukaemia cell lines

Anacardium occidentale leaves are used in folk medicine due its therapeutic properties attributed to phenolic compounds. Therefore, this study was undertaken on its hydroethanolic leaf extract (AoHE) to evaluate cytotoxicity and apoptosis induction on acute lymphoblastic leukaemia cells. Results indicated that AoHE interfered in the cell cycle progression, inducing apoptosis by activation of casp3 at lower concentrations, thence, a promising candidate for the development of new cancer drugs.

Combinatorial effects of histone deacetylase inhibitors (HDACi), vorinostat and entinostat, and adaphostin are characterized by distinct redox alterations.

Amongst the epigenetically targeted therapies, targeting of the histone deacetylases (HDACs) has yielded numerous drugs for clinical use in hematological malignancies, but none as yet for acute lymphocytic leukemia (ALL). Single agent activity of HDAC inhibitors (HDACi) has been elusive in ALL, and has prompted study of combinatorial strategies. Because several HDACi raise levels of intracellular oxidative stress, we evaluated combinations of two structurally distinct HDACi with the redox active compound adaphostin in ALL. The HDACi vorinostat and entinostat were tested in combination with adaphostin in human ALL cell lines. DNA fragmentation, caspase activation, mitochondrial disruption and levels of intracellular peroxides, superoxide and glutathione were measured in cells treated with the HDACi/adaphostin combinations. Antioxidant blockade of cell death induction and gene expression profiling of cells treated with vorinostat/adaphostin versus entinostat/adaphostin combinations were evaluated. Both combinations synergistically induced apoptotic DNA fragmentation, which was preceded by an increase in superoxide levels, a reduction in mitochondrial membrane potential, and an increase in caspase-9 activation. The antioxidant N-acetylcysteine (NAC) blocked superoxide generation and prevented reduction of mitochondrial membrane potential. NAC decreased DNA fragmentation and caspase activity in cells treated with adaphostin and vorinostat, but not in those treated with adaphostin and entinostat. Gene expression arrays revealed differential regulation of several redox genes prior to cell death induction. A redox modulatory agent, adaphostin, enhances efficacy of two HDACi, vorinostat or entinostat, but via different mechanisms indicating a point of divergence in the mechanisms of synergy between the two distinct HDACi and adaphostin.

Cytotoxicity of Pogonatum cirratum extracts against CCL-119, a human T-cell acute lymphoblastic leukaemia cell line

Cytotoxicity of Pogonatum cirratum extracts against CCL-119, a human T-cell acute lymphoblastic leukaemia cell line

Flavonoid Component Determination and Apoptotic Induction Evaluation of Houttuynia cordata Thunb Extract on Human Acute Leukemic Cells

Development of resistance to currently used drugs and side effects of several allopathic drugs have led to increased emphasis on plant materials uses as a source of medicines for a wide variety of human illnesses including leukemia. Houttuynia cordata Thunb (H.cordata), a Northern Thailand local plant and commonly known as Plucao, has various biological activities such as anti-inflammatory, anti-cancer and anti-leukemic activities. We aimed to determine active flavonoid components of H.cordata and to investigate the effect of H.cordata ethanolic extract on apoptotic induction on human acute lymphoblastic leukemia cells. In this study, we found that H.cordata ethanolic extract had total flavonoid of 231.21 ± 4.19 mg QE/g dried H.cordata. The extract analyzed by quantitative LC-MS consists of several flavonoid components including hyperin 6.35 ± 0.41, quercetin 0.34 ± 0.02, isoquercetin 1.10 ± 0.03, and rutin 0.88 ± 0.04 (%w/w). The cytotoxicity results showed dose dependent decrease in growth of Jurkat leukemic cells. Blebbing pattern of cell apoptosis was found in cells treated with H.cordata ethanolic extract for 24 and 48 hrs. Moreover, we found that the extract could substantially induce Jurkat cell death through apoptosis at both 12 and 24 hrs. In conclusion, these results indicated that H.cordata ethanolic extract which is composed of several flavonoids, possesses anti-leukemic activity through apoptotic induction in Jurkat leukemia cells.

Matrine induced G0/G1 arrest and apoptosis in human acute T-cell lymphoblastic leukemia (T-ALL) cells.

Matrine, a natural product extracted from the root of Sophora flavescens, is a promising alternative drug in different types of cancer. Here, we aimed to investigate the therapeutic effects and underlying molecular mechanisms of matrine on human acute lymphoblastic leukemia (ALL) cell line, CCRF-CEM. Cell viability and IC50 values were determined by WST-1 cell cytotoxicity assay. Cell cycle distribution and apoptosis rates were analyzed by flow cytometry. Expression patterns of 44 selected miRNAs and 44 RNAs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the Applied Biosystems 7500 Fast Real-Time PCR System. Matrine inhibited cell viability and induced apoptosis of CCRF-CEM cells in a dose-dependent manner. Cell cycle analysis demonstrated that matrine-treated CCRF-CEM cells significantly accumulated in the G0/G1 phase compared with the untreated control cells. hsa-miR-376b-3p (-37.09 fold, p = 0.008) and hsa-miR-106b-3p (-16.67 fold, p = 0.028) expressions were decreased, whereas IL6 (95.47 fold, p = 0.000011) and CDKN1A (140.03 fold, p = 0.000159) expressions were increased after matrine treatment. Our results suggest that the downregulation of hsa-miR-106b-3p leads to the upregulation of target p21 gene, CDKN1A, and plays a critical role in the cell cycle progression by arresting matrine-treated cells in the G0/G1 phase.

Prim-O-glucosylcimifugin induces cell cycle arrest and apoptosis in acute lymphoblastic leukemia cells.

Prim-O-glucosylcimifugin is a major constituent in Radix Saposhnikovia that has been long used for the treatment of pyrexia, rheumatism, and cancer in traditional Chinese medicine. However, the molecular and cellular mechanisms remain unknown regarding the therapeutic effect of prim-O-lucosylcimifugin. Here, we investigated the effects of prim-O-glucosylcimifugin on cell cycle progression and apoptosis in human acute lymphoblastic leukemia cells. Prim-O-glucosylcimifugin treatment resulted in marked increases in cell apoptosis and cell cycle arrest at the G2/M phase. Mechanistically, prim-O-glucosylcimifugin induced the degradation of β-tubulin and downregulated phosphorylated CDK1 levels, a molecular indicator in the G2/M cell cycle arrest. Furthermore, activation of caspase-3, caspase-8, and caspase-9 was involved in the prim-O-glucosylcimifugin-induced apoptosis. Our study reveals the anticancer activity of prim-O-glucosylcimifugin and the potential underlying mechanisms.

Tumor cell capture patterns around aptamer-immobilized microposts in microfluidic devices.

Circulating tumor cells (CTCs) have shown potential for cancer diagnosis and prognosis. Affinity-based CTC isolation methods have been proved to be efficient for CTC detection in clinical blood samples. One of the popular choices for affinity-based CTC isolation is to immobilize capture agents onto an array of microposts in microchannels, providing high CTC capture efficiency due to enhanced interactions between tumor cells and capture agents on the microposts. However, how the cells interact with microposts under different flow conditions and what kind of capture pattern results from the interactions have not been fully investigated; a full understanding of these interactions will help to design devices and choose experimental conditions for higher CTC capture effeciency. We report our study on their interaction and cell distribution patterns around microposts under different flow conditions. Human acute lymphoblastic leukemia cells (CCRF-CEM) were used as target cancer cells in this study, while the Sgc8 aptamer that has specific binding with CCRF-CEM cells was employed as a capture agent. We investigated the effects of flow rates and micropost shapes on the cell capture efficiency and capture patterns on microposts. While a higher flow rate decreased cell capture efficiency, we found that the capture pattern around microposts also changed, with much more cells captured in the front half of a micropost than at the back half. We also found the ratio of cells captured on microposts to the cells captured by both microposts and channel walls increased as a function of the flow rate. We compared circular microposts with an elliptical shape and found that the geometry affected the capture distribution around microposts. In addition, we have developed a theoretical model to simulate the interactions between tumor cells and micropost surfaces, and the simulation results are in agreement with our experimental observation.

Hyperosmotic stress enhances cytotoxicity of SMAC mimetics

Inhibitors of apoptosis (IAP) proteins contribute to cell death resistance in malignancies and emerged as promising targets in cancer therapy. Currently, small molecules mimicking the IAP-antagonizing activity of endogenous second mitochondria-derived activator of caspases (SMAC) are evaluated in phase 1/2 clinical trials. In cancer cells, SMAC mimetic (SM)-mediated IAP depletion induces tumor necrosis factor (TNF) secretion and simultaneously sensitizes for TNF-induced cell death. However, tumor cells lacking SM-induced autocrine TNF release survive and thus limit therapeutic efficacy. Here, we show that hyperosmotic stress boosts SM cytotoxicity in human and murine cells through hypertonicity-induced upregulation of TNF with subsequent induction of apoptosis and/or necroptosis. Hypertonicity allowed robust TNF-dependent killing in SM-treated human acute lymphoblastic leukemia cells, which under isotonic conditions resisted SM treatment due to poor SM-induced TNF secretion. Mechanistically, hypertonicity-triggered TNF release bypassed the dependency on SM-induced TNF production to execute SM cytotoxicity, effectively reducing the role of SM to TNF-sensitizing, but not necessarily TNF-inducing agents. Perspectively, these findings could extend the clinical application of SM.

Abstract 3033: The PRL family of tyrosine phosphatases as a novel drug targets in acute lymphoblastic leukemia

Abstract The aggressive and unpredictable behavior of acute lymphoblastic leukemia (ALL) presents a major clinical challenge in both the pediatric and adult setting. The development of new and better chemotherapies for this disease requires a detailed understanding of the genes and pathways that drive ALL malignancy. Phosphatases are emerging as new drug targets with important roles in leukemia progression. To determine the extent to which phosphatases play a role in ALL malignancy, we used CRISPR/Cas9 to knock out expression of 255 individual phosphatases in human T-cell acute lymphoblastic leukemia cell lines and screened for deleterious effects on cell viability. Top hits from this screen included the protein tyrosine phosphatase type IVa family, also known as the PRLs. PRL3 is genomically amplified in a subset of human T-ALL, and more than 50% of human ALL patients have significantly higher expression of PRL2 and/or PRL3, compared to normal lymphocytes, suggesting that these PRLs may play an important role in ALL malignancy. Transgenic Myc-induced ALL models in zebrafish showed that overexpression of PRL2 and PRL3 significantly shortened latency of primary and relapse ALL, enhanced leukemia stem cell self-renewal (PRL2) and prevented apoptosis after standard chemotherapy treatment (PRL3). Finally, a specific PRL inhibitor strongly induced apoptosis of PRL-expressing ALL cells in a dose-dependent manner, in both zebrafish models in vivo and human cell lines in vitro. We have identified several FDA-approved, general phosphatase inhibitors that have potent anti-PRL activity and are capable of killing ALL cells in vitro. Current work is focused on moving these inhibitors into pre-clinical testing using patient-derived xenografts. Pull-down approaches are also being used to identify the substrates of PRL phosphatase activity that are critical to ALL survival and may represent new, tractable drug targets for the treatment of ALL. &amp;lt;!–EndFragment–&amp;gt; Citation Format: Jessica S. Blackburn. The PRL family of tyrosine phosphatases as a novel drug targets in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3033. doi:10.1158/1538-7445.AM2017-3033