Apoptosis In Acute Lymphoblastic Leukemia Research Articles

Effects of PCI-32765 and Dasatinib on the Acute Lymphoblastic Leukemic Cells and Their Mechanisms

To investigate the effects of Btk inhibitor (PCI-32765) and BCR-ABL tyrosine kinase inhibitor (Dasatinib) on proliferation and apoptosis of acute lymphoblastic leukemia (ALL) cell lines (Sup-B15, RS4;11) and the possible mechanism. RS4;11 and Sup-B15 cells were treated with PCI-32765 and Dasatinib, the cell proliferation and apoptosis were detected by CCK-8, the Btk and other apoptotic proteins were detected by Western blot. PCI-32765 could inhibit the proliferation of RS4;11 and Sup-B15 cells in a dose-dependent manner, Sup-B15 cells were more sensitive to PCI-32765 than RS4;11 cells, their IC50 were 3 µmol/L and 8 µmol/L respectively, the difference between them was statistically significant (P<0.05). Dasatinib also could inhibit the proliferation of RS4;11 cells and Sup-B15 cells in a dose-dependent manner. The IC50 was 5 µmol/L and 5 nmol/L, respectively, the difference between them was statistically very significant (P<0.01), and the inhibitory effect was enhanced by the combination of Damatinib with the PCI-32765(P<0.05). The cell survival rate decreased gradually in PCI-32765 or Dasatinib alone group and the combination group at the different time-point (8, 12, 24, 36, 48 and 72 h), the 2 drugs showed a synergistic effect on cells in a time-dependent manner. After being treated with PCI-32765 and Dasatinib, the RS4;11 and Sup-B15 cells showed that cell shrinkage, increase of cytoplasmic density, nuclear pyknosis, deviation and karyorrhexis, and increase of the apoptotic cells in the combination group, while the promotive effect of low dosage dasatinib on apoptosis of RS4;11 cells was not strong. PCI-32765 and Dasatinib could decrease the expression and activity of BCR-ABL, Btk, Lyn, Src in Sup-B15 and RS4;11 cells. PCI-32765 or Dasatinib can inhibit the proliferation and induce the apoptosis of Sup-B15 and RS4;11 cells, PCI-32765 and Dasatinib displayed the synergistic effects. The possible mechanism may be related with the blocking of B cell receptor(BCR) signal pathway, thereby inhibiting the cell proliferation and promoting the cell apoptosis.

PTPN21 Plays a Role in Occurrence and Relapse of Acute Lymphoblastic Leukemia By Promoting the Proliferation of Acute Lymphoblastic Leukemic Cells Via Activating MAPK Signal Pathways

PTPN21 Plays a Role in Occurrence and Relapse of Acute Lymphoblastic Leukemia by Promoting the Proliferation of Acute Lymphoblastic Leukemic Cells via Activating MAPK Signal PathwaysAccumulating evidences have uncovered the relevance of overexpression in protein tyrosine phosphatases (PTPs) to hematologic malignancies. We analyzed 21 acute lymphoblastic leukemia (ALL) patients and 17 nonmalignant patients as control group in a pilot study. Mononuclear cells were separated from bone marrow samples of these patients via bone marrow aspiration. By real-time fluorescence quantitative PCR, we analyzed the mRNA expression of PTPN21 (tyrosine-protein phosphatase non-receptor type 21), a member of protein tyrosine phosphatases. The mRNA expression of PTPN21 in ALL group was higher than that in control group (10.86¡À2.266 vs. 6.112¡À0.8385, P£¾0.05, Figure 1A), although without significant differences. We divided 21 ALL patients into two groups: new diagnosis or relapse (61.9%, group A) and remission after chemotherapy or HSCT (38.1%, group B). The mRNA expression of PTPN21 in group A was significantly higher than that in control group (15.17¡À3.091 vs. 6.112¡À0.8385, P£¼0.01, Figure 1B) and also higher than that in group B (15.17¡À3.091 vs. 3.845¡À0.731, P£¼0.05, Figure 1B). There was no significant difference between group B and control group (3.845¡À0.731 vs. 6.112¡À0.8385, P£¾0.05, Figure 1B). These data implied that the overexpression of PTPN21 may relate to the occurrence and relapse of ALL.In order to define the specific mechanisms, we overexpressed PTPN21 in three ALL cell lines by lentiviral transfection, including Jurkat, NALM-6 and Reh cells. We assessed cell cycles by flow cytometry after cells were serum-deprived overnight and stimulated with EGF (epidermal growth factor) for 30 min. We found that cells in G0 phase dramatically decreased and cells in S/G2/M phases dramatically increased after overexpression of PTPN21 (Figure 2). We also detected apoptosis by flow cytometry and found no significant differences between overexpression group and control group. These data demonstrated that overexpression of PTPN21 promoted the proliferation of ALL cells, however didn`t affect the apoptosis of ALL cells.Next we attempted to figure out the mechanisms that PTPN21 promoted proliferation of ALL cells. We discovered the promotion of dephosphorylation of SRC residue Y527 by western blot analysis, which could stimulate SRC activity. The direct combination of PTPN21 and SRC was demonstrated by immunoprecipitation. The downstream MAPK pathways were also activated by phosphorylation. To be specific, JNK and ERK pathways were activated in Jurkat cells while P38 and ERK pathways were activated in NALM-6 cells. In addition, JNK, P38 and ERK pathways were all activated in Reh cells. In conclusion, our study revealed that PTPN21 may play a role in the occurrence and relapse of ALL by promoting the proliferation of ALL cells via activating SRC and downstream MAPK signal pathways. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The effect of miR-146a on STAT1 expression and apoptosis in acute lymphoblastic leukemia Jurkat cells.

The effect of miR-146a-dependent regulation of STAT1 on apoptosis in acute lymphoblastic leukemia (ALL) Jurkat cells was investigated. The miR-146a mimic and miR-146a inhibitor vectors were constructed in vitro, and experimental grouping was as follows: Control group (untreated Jurkat cells), empty vector group (Jurkat cells transfected with empty vector), agonist group (Jurkat cells transfected with miR-146a mimic) and the inhibitor group (Jurkat cells transfected with miR-146a inhibitor). Western blot analysis was used to observe the expression, respectively, of STAT1, p-STAT1 and Bcl-xL, and flow cytometry was used to test apoptosis in Jurkat cells. STAT1 and p-STAT1 expression in the agonist group was higher than that in the control and empty vector groups, but lower in the inhibitor group, and differences were statistically significant (P<0.05). The rate of apoptosis in the agonist group was significantly higher than that of the control group and blank vector group, and it was significantly lower in the inhibitor group (P<0.05). As a tumor suppressor, miR-146a can regulate expression of apoptosis-promoting factor STAT1, and anti-apoptosis factor Bcl-xL, and is able to promote apoptosis of ALL Jurkat cells.

Investigating Effects of Acidic pH on Proliferation, Invasion and Drug-Induced Apoptosis in Lymphoblastic Leukemia.

Some studies have shown that extracellular pH in tumors, which results in tumor progression, is less than that in normal tissues. The aim of this study was to investigate the effects of extracellular acidic pH on proliferation, invasion, and drug-induced apoptosis in acute lymphoblastic cells. The cells were cultured in different pH (pH6.6 and pH7.4) for 12days. Cell proliferation was assessed by MTT assay and cell invasion was assayed by invasion assay and gene expression analysis of MMP-9. Drug-induced apoptosis was evaluated after exposure to doxorubicin for 24hours by annexin V/PI staining and gene expression analysis of BAX pro-apoptotic protein. The results indicated the enhanced growth and invasion of leukemic cells at pH6.6 (P≤0.05). Furthermore, the cells at pH6.6 were resistant to apoptosis by doxorubicin (P≤0.05). It can be concluded that acidic pH increases the proliferation, invasion and reduces the drug-induced apoptosis in acute lymphoblastic leukemia. Extracellular acidity can influence the behavior of leukemic cells and therefore, the manipulation of extracellular liquid can be selected as a therapeutic strategy for leukemia, especially for acute lymphoblastic leukemia.

Huang Qi Huai Granules Induce Apoptosis in Acute Lymphoblastic Leukemia Cells through the Akt/FoxO1 Pathway

In recent years, a traditional Chinese medicine named Huang Qi Huai (HQH) has been frequently used in China for solid tumor therapy. However, the role of HQH on leukemia cells and its underlying mechanisms have not been elucidated. In this study, we investigated the effect of HQH on the proliferation and apoptosis of acute lymphoblastic leukemia (ALL) cell lines. Sup-B15 and Nalm-6 cells were treated with gradient doses of HQH for 24, 48 or 72 h. Cell viability was measured using a CCK8 assay and cell cycle distribution and apoptosis levels were analyzed using flow cytometry. Western blotting was used to assess the levels of proteins associated with the apoptotic pathway. The results revealed that cell survival decreased significantly with increasing concentrations of HQH. HQH induced G2 cell-cycle arrest and cell apoptosis in a dose-dependent manner. HQH inhibited phosphorylated-Akt, phosphorylated- FoxO1 and Bcl2 expression and upregulated Bim, cleaved-caspase-3 and Bax expression in a dose-dependent manner, which suggests that HQH induces the apoptosis of ALL cells via the Akt/FoxO1 pathway. HQH is a potential complementary agent for the treatment of acute lymphoblastic leukemia.

Tenovin-6-mediated inhibition of SIRT1/2 induces apoptosis in acute lymphoblastic leukemia (ALL) cells and eliminates ALL stem/progenitor cells.

BackgroundAcute lymphoblastic leukemia (ALL) is a heterogeneous group of malignant disorders derived from B- or T-cell lymphoid progenitor cells. ALL often is refractory to or relapses after treatment; thus, novel targeted therapy for ALL is urgently needed. In the present study, we initially found that the level of SIRT1, a class III histone deacetylase, was higher in primary ALL cells from patients than in peripheral blood mononuclear cells from healthy individuals. But it is not clear whether inhibition of SIRT1 by its selective small molecule inhibitor Tenovin-6 is effective against ALL cells.MethodsWe tested the effect of Tenovin-6 on ALL cell lines (REH and NALM-6) and primary cells from 41 children with ALL and 2 adult patients with ALL. The effects of Tenovin-6 on cell viability were determined by MTS assay; colony-forming assays were determined by soft agar in ALL cell lines and methylcellulose medium in normal bone marrow cells and primary ALL blast cells; cell apoptosis and cell cycling were examined by flow cytometry; the signaling pathway was determined by Western blotting; ALL stem/progenitor cells were seperated by using MACS MicroBead kit.ResultsThe results showed that Tenovin-6 treatment activated p53, potently inhibited the growth of pre-B ALL cells and primary ALL cells, and sensitized ALL cells to frontline chemotherapeutic agents etoposide and cytarabine. Tenovin-6 induced apoptosis in REH and NALM-6 cells and primary ALL cells and diminished expression of Mcl-1 and X-linked inhibitor of apoptosis protein (XIAP) in such cells. Furthermore, inhibition of SIRT1 by Tenovin-6 inhibited the Wnt/β-catenin signaling pathway and eliminated ALL stem/progenitor (CD133 + CD19-) cells.ConclusionOur results indicate that Tenovin-6 may be a promising targeted therapy for ALL and clinical trials are warranted to investigate its efficacy in ALL patients.

PI3Kdelta Inhibitor, CAL-101, De-Adheres Primary Pre-B ALL from VCAM-1 and Induces Apoptosis in Primary Pre-B ALL

BACKGROUND: Relapse remains a major problem in the treatment pre-B acute lymphoblastic leukemia (ALL). ALL cells are physically anchored in the bone marrow (BM) microenvironment through a network of adhesion molecules. Adhesion also creates intracellular signals that regulate proliferation and cell death. We have previously identified integrin α4 (α4) as a critical molecule for such cell-adhesion-mediated drug resistance (CAM-DR) in pre-B ALL. In chronic lymphocytic leukemia (CLL), α4-mediated activation of the PI3K/AKT pathway was reported. In ALL, stromal cell protection of B-lineage ALL cells has also been shown to require active Akt. However, it remains elusive how this target can be therapeutically harnessed. Therefore, we investigated if directly targeting the PI3K/AKT pathway downstream of α4 can overcome CAM-DR in pre-B ALL using a novel, FDA-approved PI3Kδ inhibitor, CAL-101METHODS: For in vitro studies, murine integrin α4 conditional knockout mouse model (α4fl/fl) oncogenically transformed with BCR-ABL1+ and patient-derived (primary) pre-B ALL cells (Philadelphia chromosome negative) co-cultured with murine calvaria-derived mesenchymal stromal (OP9) cells were used. Annexin V/7-AAD staining was used for viability determination by flow cytometry and Western Blot was used for determination of PI3Kδ and p-Akt expression. For in vivo experiments, we used a NOD/SCID IL2Rγ-/- xenograft model of primary pre-B ALL.RESULTS: Integrin α4fl/fl cells were cultured under lymphoid-skewing conditions and oncogenically transformed using BCR-ABL1 (p210). Subsequent transduction with CreERT2 or EmptyERT2 generated leukemia cells in which α4 ablation could be induced (CreERT2) or not (EmptyERT2) by addition of Tamoxifen while plated on murine VCAM-1. In the absence of a4, mouse pre-B ALL cells contain markedly reduced levels of pAKT (ser473). In a parallel experiment, we observed in primary pre-B ALL cells co-cultured with OP9 cells an increase in p-AKT compared to media-only cultured ALL cells indicating activation of the PI3K/AKT pathway upon adhesion to stroma. Under starvation conditions, primary pre-B ALL cells (LAX7R) were incubated for 4 hours without serum or stroma, and subsequently treated for 48 hours with an integrin α4 blocking antibody or with control IgG4. By Western Blot, we detected decreased levels of activated p-AKT in the cells with α4 blockade, but not with control, indicating that the AKT pathway is affected by modulation of α4.To directly target the PI3K/AKT pathway, we used CAL-101, a small molecule inhibitor highly selective for the δ isoform, which was recently FDA-approved for treatment of CLL and Non-Hodgkin Lymphoma (NHL). Previously, in primary samples from CLL patients and ALL cell lines, CAL-101 blocked PI3Kδ-AKT signaling and promoted apoptosis. However, a preclinical evaluation in primary pre-B ALL patients in vitro and in vivo are missing. For these reasons, we used CAL-101 in the following experiments as a specific PI3Kδ inhibitor in primary pre-B ALL. Expression of PI3Kδ was demonstrated by Western Blot in 8 out of 8 cases of primary pre-B ALL. After incubation of primary pre-B ALL cells with different concentrations of CAL-101 (0.1-10 µM), dose-dependent downregulation of pAkt was observed. ALL cells were plated on immobilized VCAM1 (the counter-receptor for α4) or OP9 cells and treated with vehicle control (DMSO) or CAL-101 (10 µM) for 2 days. CAL-101 de-adhered ALL cells 69.5%±0.7% from VCAM1, and only 13%±0.5% from OP-9 cells, indicating an inhibition of the of the VCAM-1 and α4 interaction. ALL cells treated with CAL-101 (10 µM) for 5 days showed a marked decrease in viability as demonstrated by AnnexinV negative and 7AAD negative staining compared to DMSO-treated controls (66.2%±1.5% vs 87.9%±1.0%, p=0.0003) indicating that mono-treatment with CAL-101-decreases viability of ALL cells. Further studies addressing whether PI3Kδ inhibition is beneficial in the setting of combination chemotherapy for ALL in vitro and in vivo are ongoing.CONCLUSION: Taken together, our data demonstrate that the AKT pathway is affected by modulation of α4. PI3Kδ inhibition with CAL-101 de-adheres pre-B ALL cells from the α4-counter-receptor VCAM1, downregulates pAkt and induces apoptosis in ALL. Data derived from further studies will determine the potential of the FDA-approved PI3Kδinhibitor CAL-101 as a novel therapy for pre-B ALL. DisclosuresWayne:MedImmune: Honoraria, Research Funding, Travel Support Other; NIH: Co-inventor on investigational products with patents, Co-inventor on investigational products with patents Other.

FOXM1 Mediates Drug-Resistance and Represents a Therapeutic Target in Pre-B Acute Lymphoblastic Leukemia

Background & Significance: Pre-B acute lymphoblastic leukemia (ALL) emerges in virtually all cases from B cell precursors that are arrested at the pre-B cell receptor checkpoint. In a gene expression survey of early B cell development, we found specific upregulation of FOXM1 at this developmental stage. FOXM1 belongs to the forkhead box transcription factor family and is a key regulator of cell growth by promoting cell cycle progression and has been implicated in progression of solid tumors. Therefore, we characterized the function and regulation of FOXM1 in normal B cell development as well as in pre-B ALL.Results: First, we verified the upregulation of FOXM1 during B cell development by qRT-PCR on sorted human and murine B cell progenitor populations. Then, we crossed Mb1-Cre tg mice to a Foxm1 conditional knockout mouse model (Foxm1fl/fl) and analyzed the early B cell populations according to the Hardy fractions. Despite the observed high expression of Foxm1 mRNA in fraction C’ and D, Foxm1 deletion did not alter B cell development. In order to investigate a potential role of FOXM1 in transformed B cells, we compared FOXM1 protein levels in patient-derived pre-B ALL samples with healthy B cells and B cell precursors and found 10-60-fold higher expression in the transformed B cell progenitors. To evaluate a potential predictive value of FOXM1 levels in patient-derived ALL samples, we measured FOXM1 mRNA levels at the time of diagnosis which strikingly correlate with risk stratification of ALL (intermediate-risk ALL n=31 vs. high risk ALL n=21; P=7.3e-5; BFM-REZ 2002). To further study the function and regulation of FOXM1, we cultured murine B cell precursors in the presence of IL7 and induced transformation with a retroviral BCR-ABL1 expression vector. BCR-ABL1 expression increased levels of FOXM1 compared to the normal IL7-dependent pre-B cells. Short-term inhibition of BCR-ABL1 did not affected protein levels of FOXM1. However, after 4 days of tyrosine kinase inhibition (TKI) treatment, FOXM1 protein levels were significantly downregulated in a dose-dependent manner. BCL2 overexpression prevented apoptosis induction by TKI but FOXM1 downregulation was retained. In addition, we found evidence that inactivation of FOXO factors by the PI3K/AKT pathway contributes to high FOXM1 expression in Ph+ ALL. Overexpression of a constitutively active form of Akt to prevent activation of FOXO factors in the presence of TKI abrogated FOXM1 downregulation. Similarly, BCR-ABL1+ ALL derived from FOXO3a knockout mice prevented TKI-mediated FOXM1 reduction. Overexpression of a constitutively active form of FOXO3a but not FOXO1 significantly reduces levels of FOXM1 expression. In line with this, we found a significant inverse correlation of FOXM1 with FOXO3A mRNA levels in Ph+ ALL patients from the ECOG E2993 trial. However, the requirement of long-term treatment indicates, that, in addition to FOXO3a activation, epigenetic regulation of the FOXM1 promoter downstream of BCR-ABL1 is required. Consistent with this finding, the FOXM1 promoter region was found to be de-methylated in a large fraction of ALL. In order to further study FOXM1 function, we transduced pre-B cells derived from Foxm1fl/fl mice with BCR-ABL1 and with an inducible ERT2-Cre vector. Deletion of Foxm1 in BCR-ABL1-driven leukemia decreases cell viability, colony formation, and proliferative capacity in vitro as well as leukemia formation in vivo. FOXM1-deleted ALL cells revealed a strikingly higher sensitivity towards TKI-treatment compared to the control cells in Imatinib dose-response curves (IC50 EV:420 nM vs IC50 Cre-ERT2: 160 nM) as well as annexin V staining. We identified the ROS scavenger Catalase as a critical target of FOXM1 in mediating this drug resistance. As potential therapeutic agents to target FOXM1, we evaluated the effects of a previously described ARF peptide and the natural occurring antibiotic Thiostrepton. Both bind FOXM1 and inhibit its function as shown by reduced mRNA expression of FOXM1 target genes (Cyclin B1, PLK1, AURKB) and induced apoptosis in ALL and prolonged survival of patient-derived ALL transplant recipient mice.Conclusion: We have identified a critical function of the transcription factor FOXM1 in mediating proliferation and drug-resistance in B cell lineage ALL, but not in normal B cell progenitors and validated FOXM1 as a therapeutic target in a large fraction of drug-resistant B cell lineage ALL. DisclosuresNo relevant conflicts of interest to declare.

Smac mimetic and glucocorticoids synergize to induce apoptosis in childhood ALL by promoting ripoptosome assembly

AbstractApoptosis resistance contributes to poor outcome in pediatric acute lymphoblastic leukemia (ALL). Here, we identify a novel synergistic combination of Smac mimetic BV6 and glucocorticoids (GCs) (ie, dexamethasone, prednisolone) to trigger apoptosis in ALL cells. BV6 and GCs similarly cooperate to induce apoptosis in patient-derived leukemia samples, underlining the clinical relevance. Importantly, BV6/dexamethasone cotreatment is significantly more effective than monotherapy to delay leukemia growth in a patient-derived xenograft model of pediatric ALL without causing additional side effects. In contrast, BV6 does not increase cytotoxicity of dexamethasone against nonmalignant peripheral blood lymphocytes, mesenchymal stromal cells, and CD34-positive hematopoietic cells. We identify a novel mechanism by showing that BV6 and dexamethasone cooperate to deplete cIAP1, cIAP2, and XIAP, thereby promoting assembly of the ripoptosome, a RIP1/FADD/caspase-8-containing complex. This complex is critical and is required for BV6/dexamethasone-induced cell death, because RIP1 knockdown reduces caspase activation, reactive oxygen species production, and cell death. Ripoptosome formation occurs independently of autocrine/paracrine loops of death receptor ligands, because blocking antibodies for TNFα, tumor necrosis factor-related apoptosis-inducing ligand, or CD95 ligand or knockdown of death receptors fail to rescue BV6/dexamethasone-induced cell death. This is the first report showing that BV6 sensitizes for GC-triggered cell death by promoting ripoptosome formation with important implications for apoptosis-targeted therapies of ALL.

A Novel Platform For The Identification Of Stromal-Derived Factors That Enhance Acute Lymphoblastic Leukemia Cell Survival In The Context Of Maintenance Chemotherapy

IntroductionChildhood B-lineage acute lymphoblastic leukemia (ALL) is the most common form of malignancy in children. ALL is highly responsive to intensive, multi-agent chemotherapy, and most children enter remission; 25% of children relapse. Furthermore, cure requires an additional two years of low intensity maintenance chemotherapy with 6-mercaptopurine (6-MP) and methotrexate. It is known that serum levels of maintenance chemotherapy drugs are not highly cytotoxic for ALL cells, and it is thought that bone marrow stromal cells (BM-SC) provide trophic signals to leukemia cells that result in increased resistance to standard therapy. If anti-apoptotic signals from stromal cells to leukemia cells were known, new molecular targets for leukemia therapy could be developed. We are developing a conceptually simple screening system that will identify stromal cell-derived molecules that support ALL cell survival in the setting of 6MP and methotrexate. Our hypothesis is based on the observation that in serum-free conditions, BMSC prevent apoptosis of primary ALL cells. We reason that if we interfere with the production of key BMSC-derived trophic factors, an increase in ALL cell apoptosis in vitro will be seen. MethodsThe system has 3 components: (1) human marrow stromal cells; (2) primary ALL cells (not established cell lines); and (3) siRNAs to knockdown candidate stromal genes. We employ a human mesenchymal stromal cell (MSC) line immortalized with a human TERT gene that has been shown to be representative of primary human stroma. 20,000 BMSC are placed in wells of a 96-well plate and treated with siRNA. 48 hours later, 30,000 primary human ALL cells are added in serum-free media. After 5 days, viable ALL cells are measured by flow cytometry. Results(1) The addition of pharmacological concentrations of 6-MP to BMSC/ALL co-cultures does not significantly increase primary ALL apoptosis. Co-cultures were established as above and 6-MP was added at 1 micromolar. ALL survival was measured at 5 days. No increase in ALL survival was seen in the 6-MP containing cultures. On unmanipulated stroma cultured without 6-MP, 1948.8 (± 397.0) viable ALL cells were recovered; on manipulated stroma in the presence of 6-MP, 1698.5 (±299.3) (p value = 0.12). (2) Interference with stromal cell protein synthesis significantly increases ALL cell apoptosis. We hypothesize that interference with new protein synthesis might reduce stromal support of ALL cells. To test this we treated BMSC for 6 hours with 25 micrograms of G418 which irreversibly blocks polypeptide synthesis. Wells were then washed with serum-free medium. Stromal cells remained viable for up to 1 week. However, ALL cells apoptosis was much higher on such treated stromal cells (32.7±18.5 viable ALL cells on G418-treated stroma versus 1948.8 ± 397.0 on unmanipulated stroma, p value< 0.001)(Figure 1). (3) Interference with global stromal cell RNA transcription significantly increased ALL cell apoptosis. Triptolide is a molecule that irreversibly inhibits RNA transcription. BMSC were treated for 6 hr with 1 microgram triptolide and washed with serum-free medium. Stromal cells remain viable for up to 1 week. ALL cell apoptosis was significantly reduced by triptolide treatment of stroma (not shown in Figure 1). The number of viable ALL cells recovered from triptolide-treated stroma was 252.3±113.2, compared to 996.6±239.2 on unmanipulated stroma (p< 0.001). (4) Knockdown of CXCL12 gene expression in stromal cells signficantly increase ALL cell apoptosis. For proof of principle, we have started with CXCL12, a gene shown in CLL to contribute to leukemia cell survival. BMSC were treated with 0.3 picomolar anti-CXCL12 siRNA for 24 hr. As measured by quantitative RT-PCR, CXCL12 gene transcription was reduced by 80%. Moreover, there was a 53.5% reduction in ALL cell viability in ALL cells co-cultured with CXCL12 siRNA-treated stroma compared to ALL cells co-cultured with untreated stroma (p value< 0.001) (Figure 1). [Display omitted] ConclusionsThis BMSC cell/primary ALL cell system can be used to identify stromal cell genes that prevent apoptosis of primary ALL cells. The simple system can be scaled up to allow high throughput screening of candidate genes that have antagonistic or additive effects to maintenance chemotherapy drugs. Disclosures:No relevant conflicts of interest to declare.

Thymoquinone Induces Mitochondria-Mediated Apoptosis in Acute Lymphoblastic Leukaemia in Vitro

There has been a growing interest in naturally occurring compounds from traditional medicine with anti-cancer potential. Nigella sativa (black seed) is one of the most widely studied plants. This annual herb grows in countries bordering the Mediterranean Sea and India. Thymoquinone (TQ) is an active ingredient isolated from Nigella sativa. The anti-cancer effect of TQ, via the induction of apoptosis resulting from mitochondrial dysfunction, was assessed in an acute lymphocyte leukemic cell line (CEMss) with an IC50 of 1.5 µg/mL. A significant increase in chromatin condensation in the cell nucleus was observed using fluorescence analysis. The apoptosis was then confirmed by Annexin V and an increased number of cellular DNA breaks in treated cells were observed as a DNA ladder. Treatment of CEMss cells with TQ encouraged apoptosis with cell death-transducing signals by a down-regulation of Bcl-2 and up-regulation of Bax. Moreover, the significant generation of cellular ROS, HSP70 and activation of caspases 3 and 8 were also observed in the treated cells. The mitochondrial apoptosis was clearly associated with the S phase cell cycle arrest. In conclusion, the results from the current study indicated that TQ could be a promising agent for the treatment of leukemia.

Modulation of Leukemic Stem Cell Self-Renewal and Cell Fate Decisions by Inhibition of Hedgehog Signalling in Human Acute Lymphoblastic Leukemia (ALL).

AbstractAbstract 2578The Hedgehog (Hh) pathway plays a functional role in embryonic development and promotes tumorigenesis in a diversity of cancers. Constitutive activation of Smo, an essential component of the Hh pathway, augments stem cell number and accelerates disease in BCR-ABL positive CML, whereas loss of Smo depletes CML stem cells by inhibition of self-renewal. Phase I clinical trials using Hh inhibitors have started in BCR-ABL pos ALL and CML. The role of Hh signalling on stem cell behaviour in BCR-ABL neg ALL has not yet been examined. The phenotype of leukemic stem cells (LSCs) and the target cells for transformation in ALL are controversial, but only a small subpopulation of cells seem to act as LSCs. These cells may be the most relevant targets for treatment regimens for compounds that interfere with self-renewal programs and that provide promising therapeutic options for improving treatment of adult ALL.Aims of the study are characterization of different genetically and phenotipically defined ALLs, using our twelve patient derived long term cultures (LTCs), according to their biologic behaviour including leukemia initiating capacity, assessment of the impact of Hh inhibition on proliferation, apoptosis and clonogenic capacity and LSC function and dissection of the role of different components of the Hh signalling pathway on cell fate decisions by means of single cell video microscopy. These studies are anticipated to yield information on the therapeutic potential of modulation of Hh signalling in both BCR-ABL pos and neg ALL and the potential value of combination therapy.As models of BCR-ABL pos and neg leukemias we used serum-, cytokine- and stroma-free long term cultures of primary ALL blasts. Clonogenic growth of ALL cells was assessed in semi solid methylcellulose based media. Cell subpopulations were isolated on the basis of CD20, CD34 and CD38 expression via FACS based sorting (BD FACS Aria). Cell proliferation was measured using XTT assays and Annexin V and 7 AAD FACS staining were used to quantitate apoptosis. Quantitative RT PCR of Hh signalling pathway components using predeveloped Taqman assays (Applied). Single cell video tracking to determine cell fate decisions was performed as previously described (Rieger et all, Science 2009), adapted to facilitate the analysis of ALL LTCs. Two novel Smo inhibitors being currently in clinical testing, LDE225 and BMS833923 were kindly provided by Novartis and Bristol Myers Squibb.Results: The expression pattern of surface markers varied profoundly between the different LTCs studied. In preliminary experiments designed to identify functionally distinct subpopulations of long term cultured ALL blasts, cells were isolated to greater than 90% purity based on CD20, CD34 and CD38 expression. With the exception of CD34 positive cells, the surface marker distribution rapidly reverted to an identical pattern as determined prior to culture in three cell lines studied. In two ALL LTCs, CD34 expression was associated with slower proliferation. All three cell lines displayed clonogenic capacity ranging from 0,25% to 8% and are able to engraft in NSG mice.Analysis of Hh Signalling in ALL LTCs by RT PCR demonstrated expression of Shh, Ptch, Smo, and the transcription factors Gli 1 + 3, indicating active Hh signalling in ALL. Interestingly the transcription factor Gli 2 was not expressed, the functional relevance of which remains as yet unclear. The Hh inhibitors LDE225 and BMS833923 (0,01μM to 5μM) showed no dose dependent effect on inhibition of proliferation or induction of apoptosis in ALL LTCs.In conclusion we found evidence of Hh activation in both BCR-ABL pos and neg LTC ALL cells. No impact of Smo inhibition on proliferation and apoptosis was observed in response to the Smo inhibitors LDE225 and BMS833923, consistent with the hypothesis that Hh signalling in these cells may affect primarily self-renewal mechanisms. Single cell imaging of ALL LTCs has been successfully established for up to nine days of culture and will be applied to the testing of Hh modulation on cell fate decisions. Disclosures:No relevant conflicts of interest to declare.

A new development of triterpene acid‐containing extracts from Viscum album L. displays synergistic induction of apoptosis in acute lymphoblastic leukaemia

Aqueous Viscum album L. extracts are widely used for anti-cancer therapies. Due to their low solubility, triterpenes (which are known to act on cancers), do not occur in aqueous extracts in significant amounts. Using cyclodextrins, we have found it possible to solubilize mistletoe triterpene acids and to determine their effects on acute lymphoblastic leukaemia (ALL) in vitro and in vivo. A C.B-17/SCID model of pre-B ALL (NALM-6) was used to test efficacy and mechanisms of treatment with lectin- and triterpene acid containing preparations in vivo. Cytotoxicity of increasing concentrations of V. album L. preparations was assessed in vitro. Apoptosis was determined using mitochondrial membrane potential measurements, annexin V/PI, western blot analyses and caspase inhibitor assays. Solubilized triterpene acid- or lectin-containing V. album L. extracts inhibited cell proliferation and demonstrated cytotoxic properties in vitro. Annexin V/PI and mitochondrial membrane potential assays indicated that dose-dependent induction of apoptosis was the main mechanism. Combination (viscumTT) of lectin- (viscum) and triterpene-containing (TT) extracts resulted in greatest induction of apoptosis. Furthermore, caspase activity demonstrated that these extracts were able to induce apoptosis through both caspase-8 and -9 dependent pathways. In vivo experimentation showed that treatment of mice with viscumTT combination prolonged mean survival to 50.5 days compared to 39.3 days in the phosphate-buffered saline group. Here for the first time, we have demonstrated that either solubilized triterpene acids or lectins and combinations thereof, induce dose-dependent apoptosis in the ALL cell line NALM-6 via caspase-8 and -9 dependent pathways.

Modeling the Mechanism of GR/c-Jun/Erg Crosstalk in Apoptosis of Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is one of the most common forms of malignancy that occurs in lymphoid progenitor cells, particularly in children. Synthetic steroid hormones glucocorticoids (GCs) are widely used as part of the ALL treatment regimens due to their apoptotic function, but their use also brings about various side effects and drug resistance. The identification of the molecular differences between the GCs responsive and resistant cells therefore are essential to decipher such complexity and can be used to improve therapy. However, the emerging picture is complicated as the activities of genes and proteins involved are controlled by multiple factors. By adopting the systems biology framework to address this issue, we here integrated the available knowledge together with experimental data by building a series of mathematical models. This rationale enabled us to unravel molecular interactions involving c-Jun in GC induced apoptosis and identify Ets-related gene (Erg) as potential biomarker of GC resistance. The results revealed an alternative possible mechanism where c-Jun may be an indirect GR target that is controlled via an upstream repressor protein. The models also highlight the importance of Erg for GR function, particularly in GC sensitive C7 cells where Erg directly regulates GR in agreement with our previous experimental results. Our models describe potential GR-controlled molecular mechanisms of c-Jun/Bim and Erg regulation. We also demonstrate the importance of using a systematic approach to translate human disease processes into computational models in order to derive information-driven new hypotheses.

Genistein inhibited proliferation and induced apoptosis in acute lymphoblastic leukemia, lymphoma and multiple myeloma cells in vitro

Genistein is one of the major isoflavones in soy products. It has been reported that genistein has apoptotic effects on certain hematological malignancies. However, so far there have been no completely comparative studies of the effect of genistein on malignant hematological diseases, especially multiple myeloma. We investigated genistein's inhibitory effect on the growth of acute lymphoblastic leukemia (RS4;11 and CEM), lymphoma (Toledo and GA10) and multiple myeloma (OPM-2 and U266) cell lines in vitro. We observed that genistein dose- and time-dependently inhibited proliferation of these cells. The cell line sensitivity to genistein treatment based on the 50% inhibitory concentration (IC50) values in decreasing order of toxicity was found to be as follows: RS4;11 (4.89 ± 4.28 μM) > GA10 (13.08 ± 3.49 μM) > Toledo (16.94 ± 3.89 μM) > CEM (17.31 ± 0.72 μM) > OPM-2 (46.76 ± 2.26 μM) > U266 (128.82 ± 1.90 μM). The mechanism of growth inhibition was through induction of apoptosis and cell cycle arrest. The concomitant altered expression of apoptosis pathway proteins and cell cycle modulators (caspases 9, 3, 7, PARP [poly(ADP-ribose) polymerase], cIAP1 [inhibitor of apoptosis protein 1], Bcl-2 and cyclin B1) were observed by Western blot and real-time polymerase chain reaction (PCR) analyses. In addition, some malignancy-related embryologic pathway proteins, e.g. Notch1 and Gli1, were modulated by genistein treatment in sensitive cell lines.

Borrelidin, a small molecule nitrile-containing macrolide inhibitor of threonyl-tRNA synthetase, is a potent inducer of apoptosis in acute lymphoblastic leukemia

Due to the poor prognosis and limited therapeutic options for adult patients with acute lymphoblastic leukemia (ALL), development of novel therapies is much needed to prolong patient survival and increase the efficacy of their treatment. Malignant T cells need high levels of nutrients to maintain their proliferation rate. Borrelidin, a small molecule nitrile-containing macrolide, is an inhibitor of bacterial and eukaryal threonyl-tRNA synthetase. Borrelidin-mediated inhibition of aminoacyl-tRNA synthesis, leads to an induction in the levels of uncharged tRNA, nutritional stress and ultimately inhibition of protein synthesis. The aim of the present study was to investigate whether borrelidin treatment inhibits the proliferation of malignant ALL cell lines, Jurkat and CEM cells, and study the mechanism by which this drug acts. Our results show that borrelidin was able to potently inhibit the proliferation of ALL cell lines with a half maximal inhibitory concentration of 50ng/ml. Borrelidin showed a greater inhibitory effect on ALL cell lines compared to primary fibroblasts. Flow cytometry and western blot analysis indicated that borrelidin was able to increase the level of apoptosis and cause G(1) arrest in ALL cell lines. Activation of the general control nonderepressible-2 (GCN2) kinase stress responsive pathway and induction of CHOP protein was significantly higher in ALL cell lines treated with borrelidin. These findings collectively suggest for the first time that borrelidin targets ALL cell lines by inducing apoptosis and mediating G(1) arrest and that borrelidin treatment in ALL cell lines is correlated with activation of the GCN2 kinase pathway.

Degradation of MDM2 by the Interaction between Berberine and DAXX Leads to Potent Apoptosis in MDM2-Overexpressing Cancer Cells

Berberine, a natural product derived from a plant used in Chinese herbal medicine, is reported to exhibit anticancer effects; however, its mechanism of action is not clearly defined. Herein, we demonstrate that berberine induces apoptosis in acute lymphoblastic leukemia (ALL) cells by downregulating the MDM2 oncoprotein. The proapoptotic effects of berberine were closely associated with both the MDM2 expression levels and p53 status of a set of ALL cell lines. The most potent apoptosis was induced by berberine in ALL cells with both MDM2 overexpression and a wild-type (wt)-p53, whereas no proapoptotic effect was detected in ALL cells that were negative for MDM2 and wt-p53. In contrast to the conventional chemotherapeutic drug doxorubicin, which induces p53 activation and a subsequent upregulation of MDM2, berberine strongly induced persistent downregulation of MDM2 followed by a steady-state activation of p53. We discovered that downregulation of MDM2 in ALL cells by berberine occurred at a posttranslational level through modulation of death domain-associated protein (DAXX), which disrupted the MDM2-DAXX-HAUSP interactions and thereby promoted MDM2 self-ubiquitination and degradation. Given that MDM2-overexpressing cancer cells are commonly chemoresistant, our findings suggest that this naturally derived agent may have a highly useful role in the treatment of cancer patients with refractory disease.

Berberine Induces Apoptosis In Acute Lymphoblastic Leukemia Cells by Demethylation of RASSF1A and Self-Ubiquitination of MDM2.

AbstractAbstract 3627Berberine, an isoquinoline alkaloid derived from a plant used in Chinese herbal medicine, is reported to exhibit anti-proliferative and pro-apoptotic effects on human cancer; however, its mechanism of action is not clearly defined. Herein, we demonstrate that berberine induces apoptosis in acute lymphoblastic leukemia (ALL) cells by demethylation of the tumor suppressor RASSF1A, which results in self-ubiquitination and degradation of the oncoprotein MDM2. In contrast to the conventional chemotherapeutic drug doxorubicin, which directly induced p53 activation and a subsequent upregulation of MDM2, berberine strongly induced persistent downregulation of MDM2 followed by a steady-state activation of p53, which resulted in apoptosis of ALL cells tested, even if they were doxorubicin-resistant. Our investigation discovered that berberine but not doxorubicin can induce demethylation of RASSF1A in RASSF1A-methylated ALL. The result of RASSF1A demethylation was the increased expression of the tumor suppressor, which disrupted the MDM2-DAXX-HAUSP complex, thereby promoting MDM2 self-ubiquitination and subsequent MDM2 degradation. Consistent with this finding, the most potent apoptosis was induced by berberine in wild-type p53 ALL cells with both RASSF1A methylation and MDM2 overexpression, while no pro-apoptotic effect was detected in berberine-treated RASSF1A normal ALL cells that had no or low level of MDM2 expression. Given that ALL cells with RASSF1A methylation and MDM2 overexpression are commonly chemotherapy-resistant our finding that these cells can be potently killed by berberine through effective induction of RASSF1A and inhibition of MDM2 suggests that this naturally-derived agent may have a highly useful role in the treatment of patients with refractory ALL. Disclosures:No relevant conflicts of interest to declare.

A New, Lineage Specific, Autoup-Regulation Mechanism for Human Glucocorticoid Receptor Gene Expression in 697 Pre-B-Acute Lymphoblastic Leukemia Cells

Glucocorticoid (GC) steroid hormones induce apoptosis in acute lymphoblastic leukemia (ALL). Autoup-regulation of human GC receptor (hGR) levels is associated with sensitivity to GC-mediated apoptosis. Among the major hGR promoters expressed in 697 pre-B-ALL cells (1A, 1B, 1C, and 1D), only promoters 1C and 1D are selectively activated by the hormone. Promoter 1B is unresponsive, and promoter 1A is down-regulated by dexamethasone (Dex) in 697 cells, whereas they are both up-regulated in CEM-C7 T-ALL cells. Autoup-regulation of promoter 1C and 1D in 697 cells requires sequences containing GC response units (GRUs) (1C GRU, -2915/-2956; 1D GRU, -4525/-4559) that were identified previously in CEM-C7 cells. These GRUs potentially bind GR, c-myeloblastosis (c-Myb), and E-twenty six (Ets) proteins; 697 cells express high levels of c-Myb protein, as well as the E-twenty six family protein members, PU.1 and Spi-B. Dex treatment in 697 cells elevates the expression of c-Myb and decreases levels of both Spi-B and PU.1. Chromatin immunoprecipitation assays revealed the specific recruitment of GR, c-Myb, and cAMP response element-binding protein binding protein to the 1C and 1D GRUs upon Dex treatment, correlating to observed autoup-regulated activity in these two promoters. These data suggest a hormone activated, lineage-specific mechanism to control the autoup-regulation of hGR gene expression in 697 pre-B-ALL cells via steroid-mediated changes in GR coregulator expression. These findings may be helpful in understanding the mechanism that determines the sensitivity of B-ALL leukemia cells to hormone-induced apoptosis.

FK506 Binding Proteins as Targets in Anticancer Therapy

FK506 binding proteins (FKBPs) are the intracellular ligands of FK506 and rapamycin, two natural compounds with powerful and clinically efficient immunosuppressive activity. In recent decades, a relevant role for immunosuppressants as anticancer agents has emerged. Especially, rapamycin and its derivatives are used, with successful results, across a variety of tumors. Of note, rapamycin and FK506 bind to FKBP12, and the resulting complexes interfere with distinct intracellular signaling pathways driven, respectively, by the mammalian target of rapamycin and calcineurin phosphatase. These pathways are related to T-cell activation and growth. Hyperactivation of the mammalian target of rapamycin (mTOR), particularly in cancers that have lost the tumor suppressor gene PTEN, plays an important pathogenetic role in tumor transformation and growth. The signaling pathway involving calcineurin and nuclear factors of activated T-lymphocytes is also involved in the pathogenesis of different cancer types and in tumor metastasis, providing a rationale for use of FK506 in anticancer therapy. Recent studies have focused on FKBPs in apoptosis regulation: Targeting of FKBP12 promotes apoptosis in chronic lymphocytic leukemia, FKBP38 knockdown sensitizes hepatoma cells to apoptosis, and FKBP51 silencing overcomes resistance to apoptosis in acute lymphoblastic leukemia, prostate cancer, melanoma, and glioma. Interestingly, derivatives of FK506 that have the same FKBP12-binding properties as FK506 but lack functional immunosuppressant activity, exert the same apoptotic effect as FK506 in chronic lymphocytic leukemia.These findings suggest that a direct FKBP inhibition represents a further mechanism of immunosuppressants.' anticancer activity. In this review, we focus on the role of FKBP members in apoptosis control and summarize the data on the antitumor effect of selective targeting of FKBP.