Combining RAD001 (Everolimus) with Proteasome Inhibitors Bortezomib (Velcade) or MG132 Significantly Enhances Pre-B ALL Cell Death in Vitro.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2743-2743
Author(s):  
Philip O. Saunders ◽  
Kenneth F Bradstock ◽  
Linda J. Bendall
Keyword(s):  
Cell Death ◽  
Lymphoblastic Leukemia ◽  
Significant Proportion ◽  
Receptor Activation ◽  
Jnk Pathway ◽  
Dna Damaging Agents ◽  
Pathway Activation ◽  
Wide Range ◽  
Jnk Activation

Abstract Abstract 2743 Poster Board II-719 Mammalian target of rapamycin (mTOR) inhibitors have shown potential as novel therapeutic agents with efficacy against a wide range of tumors including precursor-B acute lymphoblastic leukemia (pre-B ALL). We have previously reported RAD001 (16μM) induces JNK pathway activation in pre-B ALL cells and that combining RAD001 with DNA damaging agents in vitro significantly enhanced JNK dependent death, via a caspase dependent mechanism. We sought to evaluate agents, which may favor JNK activation and promote JNK dependent cell death in pre-B ALL cells. Bortezomib and MG132 have both been reported to activate the JNK pathway via death receptor activation, with reported efficacy in pre-B ALL. Consistent with the literature we observed enhanced JNK pathway activation in pre-B ALL cells treated with bortezomib. Analysis of annexin V and 7AAD expression by flow cytometry utilizing JNK inhibitor SP600125 (5μM) showed that a significant proportion of pre-B ALL cell death observed with bortezomib was JNK dependent. Combining RAD001 (4–16μM) with bortezomib in vitro (10–20nM) significantly enhanced cell death in pre-B ALL cell lines and patient cases at 24 hours. This observation was supported by equivalent observations combining MG132 (250–500nM) with RAD001 (8–16μM). The degree of enhanced killing was greater than that achieved combining RAD001 (16μM) with DNA damaging agents. Enhanced killing was also achieved at a significantly lower dose of RAD001 relative to combination therapy with DNA damage. Utilizing JNK inhibitor SP600125 (5μM) we determined that a significant proportion of enhanced killing was JNK dependent. In conclusion we have identified two novel and clinically available agents which when combined can significantly enhance pre-B ALL cell death. Our observations suggest combining agents which induce JNK activation has the potential to enhance clinical responses in pre-B ALL, particularly for patients with advanced or relapsed disease, for whom treatment with cytotoxic chemotherapy offers little hope of improved survival. In vivo studies will provide further insight into this promising strategy. Disclosures: No relevant conflicts of interest to declare.

The JNK Pathway Is a Significant Determinant of Sensitivity to DNA Damaging Agents in Pre-B ALL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3786-3786
Author(s):  
Philip O. Saunders ◽  
Kenneth F Bradstock ◽  
Linda J. Bendall
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Death ◽  
Dna Damage Response ◽  
Genotoxic Stress ◽  
Jnk Pathway ◽  
Dna Damaging Agents ◽  
Pathway Activation ◽  
Damage Response ◽  
Jnk Activation

Abstract Abstract 3786 Poster Board III-722 The JNK pathway is reported to facilitate AP1 binding and promote apoptosis depending on cell type and environmental conditions. We have previously reported RAD001 (16μM) induces JNK pathway activation in pre-B ALL cells. We sought to evaluate the impact of changes in JNK pathway activation on pre-B ALL viability in vitro. Using JNK inhibitor SP600125 titrated to inhibit c-Jun activation, we determined that cell death in pre-B ALL cells treated with RAD001 (16μM) alone was not JNK dependent. In contrast, combining RAD001 (16μM) with DNA damaging agents significantly enhanced JNK dependent death. This difference indicates that additional factors, including genotoxic stress, are required for JNK activation to induce pre-B ALL cell death. The JNK pathway is reported to suppress transcriptional activation of key mediators of the DNA damage response. We observed that JNK activation in cells treated with RAD001 (16μM) and DNA damaging agents was associated with suppression of p53 and p21 relative to DNA damage alone. This result was supported by the observation of enhanced p53 and p21 expression in pre-B ALL cells treated with DNA damaging agents in the presence of the JNK inhibitor SP600125. Analysis of DNA content and proliferation antigen expression in pre-B ALL cells treated with RAD001 (16μM) and DNA damaging agents revealed JNK activation was associated with a significant increase in the proportion of cells in S phase, relative to DNA damage alone, which caused a G1 and G2 cell cycle arrest. Further evidence that the JNK pathway impacts on the DNA damage response was provided by the observation that pre-B ALL cells treated with DNA damaging agents and JNK inhibitor SP600125 demonstrated reduced PCNA expression at G1 and G2 and reduced expression of mitotic antigen phospho-Histone–H3. This is consistent with enhanced regulation at G1-S and G2-M checkpoints. The results indicate changes in JNK pathway activation impact on the cell cycle response to DNA damage. In conclusion we have identified that the JNK pathway has a significant impact on the sensitivity of pre-B ALL cells to DNA damaging agents. JNK activation in the presence of genotoxic stress significantly enhanced pre-B ALL cell death, associated with suppression of key mediators of the DNA damage response, p53 and p21. We found that changes in JNK activation altered the cell cycle response to DNA damage. Further study is required to determine if changes in cell cycle regulation in the presence of DNA damage is causal to JNK dependent cell death. Additional studies to identify intracellular signal pathways which facilitate JNK dependent cell death are warranted. Our observations suggest combining agents which induce JNK activation with conventional chemotherapy or selected novel agents has the potential to enhance clinical responses in pre-B ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Protein Phosphatase 4 Promotes Hepatocyte Lipoapoptosis by Regulating RAC1/MLK3/JNK Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Xiuqing Huang ◽  
Guang Yang ◽  
Li Zhao ◽  
Huiping Yuan ◽  
Hao Chen ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Hepatic Insulin Resistance ◽  
Jnk Pathway ◽  
Pathway Activation ◽  
Jnk Signaling Pathway ◽  
Induced Apoptosis ◽  
Jnk Activation ◽  
First Time

Lipotoxicity-induced apoptosis, also referred to as lipoapoptosis, is one of the important initial factors promoting the progression from hepatosteatosis to nonalcoholic steatohepatitis (NASH). Saturated free fatty acids (SFAs), which are increased significantly in NASH, are directly hepatotoxic which induce hepatocyte lipoapoptosis. Previously, we reported that protein phosphatase 4 (PP4) was a novel regulator of hepatic insulin resistance and lipid metabolism, but its role in hepatic lipoapoptosis remains unexplored. In this study, we found out that PP4 was upregulated in the livers of western diet-fed-induced NASH mice and SFA-treated murine primary hepatocytes and HepG2 cells. In addition, we found for the first time that suppression of PP4 decreased SFA-induced JNK activation and expression of key modulators of hepatocyte lipoapoptosis including p53-upregulated modulator of apoptosis (PUMA) and Bcl-2-interacting mediator (Bim) and reduced hepatocyte lipoapoptosis level as well both in vitro and in vivo. Further study revealed that PP4 induced JNK activation and lipoapoptosis-related protein expression by regulating the RAC1/MLK3 pathway instead of the PERK/CHOP pathway. The effects of palmitate-treated and PP4-induced lipoapoptosis pathway activation were largely abolished by RAC1 inhibition. Moreover, we identified that PP4 interacted with RAC1 and regulated GTPase activity of RAC1. In conclusion, these results demonstrated that PP4 was a novel regulator of hepatocyte lipoapoptosis and mediated hepatocyte lipoapoptosis by regulating the RAC1/MLK3/JNK signaling pathway. Our finding provided new insights into the mechanisms of this process.

scholarly journals Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

2021 ◽  
Vol 22 (15) ◽  
pp. 7906
Author(s):  
Alexey A. Komissarov ◽  
Maria A. Karaseva ◽  
Marina P. Roschina ◽  
Andrey V. Shubin ◽  
Nataliya A. Lunina ◽  
...  
Keyword(s):  
Cell Death ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Proteolytic Enzymes ◽  
Morphological Changes ◽  
Human Cells ◽  
Mitochondrial Potential ◽  
3C Protease ◽  
Wide Range

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

scholarly journals ASK1-P38 Pathway is Important for Anoikis Induced by Microtubule-Targeting Aryl Chloroethylureas

2010 ◽  
Vol 13 (2) ◽  
pp. 175 ◽  
Author(s):  
Jessica S Fortin ◽  
Alexandre Patenaude ◽  
Rena G Deschesnes ◽  
Marie-France Côté ◽  
Eric Petitclerc ◽  
...  
Keyword(s):  
Cell Death ◽  
Focal Adhesion ◽  
Mapk Signaling ◽  
Pathway Activation ◽  
Differential Cell ◽  
Cell Death Mechanisms ◽  
Jnk Activation ◽  
Cytotoxic Mechanism ◽  
P38 Pathway ◽  
Sustained Increase

PURPOSE. We investigated the involvement of MAPK signaling in the cell death mechanisms of classical microtubule interfering agents (MIA) and aryl-3-(2-chloroethyl)ureas (CEU) acting as antimitotics, along with CEU that don’t affect directly microtubules (non-MIA CEU). METHODS. To ascertain the activated signaling pathway profile of MIA and non-MIA CEU, Western blot, immunoprecipitation and transfection experiments were performed. RESULTS. Non-MIA CEU do not activate p38, as opposed to MIA, and the extent of ERK and JNK activation is lower than in response to MIA. The effect of MIA and non-MIA CEU on focal adhesion associated protein was also studied; MIA were shown to induce focal adhesion dismantlement associated with a sustained increase in paxillin phosphorylation and FAK cleavage, as opposed to non-MIA CEU. In addition, bcl-2 phosphorylation and AKT cleavage, induced by all MIA tested, was not observed in response to non-MIA CEU further emphasizing the differential cell death mechanisms induced by MIA and non-MIA CEU. Pharmacologic and genetic approaches emphasize that the ASK1-p38 pathway activation contributes to the cytotoxic mechanism of MIA, in contrast to non-MIA CEU. ASK1-p38 is important for increased paxillin phosphorylation and FAK cleavage, suggesting that ASK-1-p38 is an upstream event of FA structure dismantlement induced by MIA. Moreover, the endogen inhibitor of ASK-1, thioredoxin, is released from ASK-1 in response to MIA as opposed to non-MIA CEU. CONCLUSION. Our study supports that ASK1-p38 activation is an important signaling event, induced by MIA, which impairs focal adhesion structure and induces anchorage-dependent apoptosis or anoikis.

scholarly journals cdc37 is essential for JNK pathway activation and wound closure in Drosophila

2019 ◽  
Vol 30 (21) ◽  
pp. 2651-2658
Author(s):  
Chan-wool Lee ◽  
Young-Chang Kwon ◽  
Youngbin Lee ◽  
Min-Yoon Park ◽  
Kwang-Min Choe
Keyword(s):  
Cell Growth ◽  
Wound Closure ◽  
Cell Growth And Migration ◽  
Jnk Pathway ◽  
Protein Levels ◽  
Pathway Activation ◽  
And Migration ◽  
The Stability ◽  
Jnk Activation

Wound closure in the Drosophila larval epidermis mainly involves nonproliferative, endocyling epithelial cells. Consequently, it is largely mediated by cell growth and migration. We discovered that both cell growth and migration in Drosophila require the cochaperone-encoding gene cdc37. Larvae lacking cdc37 in the epidermis failed to close wounds, and the cells of the epidermis failed to change cell shape and polarize. Likewise, wound-induced cell growth was significantly reduced, and correlated with a reduction in the size of the cell nucleus. The c-Jun N-terminal kinase (JNK) pathway, which is essential for wound closure, was not typically activated in injured cdc37 knockdown larvae. In addition, JNK, Hep, Mkk4, and Tak1 protein levels were reduced, consistent with previous reports showing that Cdc37 is important for the stability of various client kinases. Protein levels of the integrin β subunit and its wound-induced protein expression were also reduced, reflecting the disruption of JNK activation, which is crucial for expression of integrin β during wound closure. These results are consistent with a role of Cdc37 in maintaining the stability of the JNK pathway kinases, thus mediating cell growth and migration during Drosophila wound healing.

Effective Induction of Apoptosis by Mistletoe Plant Extracts in an Acute Lymphoblastic Leukemia Model.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1880-1880
Author(s):  
Georg Seifert ◽  
Patrick Jesse ◽  
Aram Prokop ◽  
Tobias Reindl ◽  
Stephan Lobitz ◽  
...  
Keyword(s):  
Cell Death ◽  
Body Weight ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
The Body ◽  
Induction Of Apoptosis ◽  
First Time ◽  
Over Time

Abstract Mistletoe (Viscum album) is one of the most used alternative cancer therapies applied as monotherapy or in combination with conventional therapies. Anti-tumor effects of mistletoe (MT) extracts were related to cytostatic and immunomodulatory effects observed in vitro. Aqueous MT extracts contain the three mistletoe lectins I, II and III as one predominant group of biologically active agents. The MT lectins inhibit protein biosynthesis by inactivating the 60S ribosomal subunit. Mistletoe lectin-I (ML-I) is one important apoptosis inducing compound. It is a heterodimer that consists of a cytotoxic A-chain (ribosome inactivating protein, RIP type 1) linked by a carbohydrate binding B-chain for cellular lectin uptake. However, although MT is widely used, there is a lack of scientific preclinical and clinical data. Here, we describe for the first time efficacy and mechanism of MT extracts against lymphoblastic leukemia in vitro and in vivo. For this purpose, we first investigated both the cytotoxic effect and mechanism of action of two standardized aqueous MT extracts (MT obtained from fir trees (MT-A); MT obtained from pine trees (MT-P)) and isolated ML-I, in three human acute lymphoblastic leukemia (ALL) cell lines (NALM-6, sup-B-15 and REH). MT-A, MT-P and ML-I clearly inhibited cell proliferation as determined by LDH reslease assays at very low concentrations (ML-I LD50 from 0,05 ng/ml to 10 ng/ml depending on the host tree) with MT-P being the most cytotoxic extract. The mechanism of cell death was determined by DNA-fragmentation assays. These indicated dose dependent induction of apoptosis as the main mechanism of cell death. Finally, we evaluated the efficacy of MT-A and MT-P in an in vivo SCID-model of pre-B ALL (NALM-6). For this purpose, mice (n=8/group) were injected i.v. with 1 × 106NALM6 cells and treated by intraperitoneal injections four times per week for 3 weeks (day 1–4; 7–11; 14–18) at varying doses (1, 5 and 50 mg/Kg (plant weight/body weight)). Mice (n=8) treated with PBS and cyclophosphamide (100 mg/kg, once on day 1) were used as negative and positive controls, respectively. Toxicity, peripheral blood counts, bodyweight and survival was determined over time. Interestingly, both MT extracts in all tested concentrations significantly improved survival (up to 55,4 days) in contrast to controls (34,6 days). Furthermore, no hematologic side effects were observed from this treatment as indicated by completely stable blood counts. Also the body weight of treated animals remained stable over time indicating a complete absence of systemic toxicity in the selected dose range. In summary, we demonstrate for the first time efficacy and mechanism of MT extracts against ALL in vitro and in vivo and hereby provide an important base line for the design of clinical trials with these compounds.

CpG Oligonucleotides Induce Anti-Leukemia Activity in a Syngeneic Murine Model of Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2830-2830
Author(s):  
Alix E. Seif ◽  
Marlo D. Bruno ◽  
Junior Hall ◽  
Valerie I. Brown ◽  
Stephan A. Grupp ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Nk Cells ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Intensive Therapy ◽  
Significant Proportion ◽  
Cpg Odn

Abstract Acute lymphoblastic leukemia (ALL) accounts for 80% of all pediatric leukemias and is the most common form of childhood cancer. While most children with ALL are cured by current therapies, refractory and relapsed ALL comprise a significant proportion of all pediatric cancers. Furthermore, nearly half of all ALL diagnoses occur in adults, who carry a much poorer prognosis, with the majority dying of relapsed disease. Relapsed ALL generally requires intensive therapy with significant associated morbidity and mortality. Development of novel therapies is essential to improving outcomes. DNA oligodeoxynucleotides containing CpG motifs (CpG ODN) stimulate anti-tumor immune activity via Toll-like receptor 9 (TLR9) activation and are currently in clinical trials for a variety of solid tumors. We have previously reported that CpG ODN stimulation alters antigen presentation by human ALL cells, enhancing allogeneic Th1 responses. In addition, we have shown that CpG ODN administration in vivo reduces the leukemic burden of primary human ALL xenografts in Nod-SCID mice, and that this activity is mediated in part by NK cells. To further the development of CpG ODN as a novel therapeutic agent for ALL, we have investigated the induction of anti-ALL activity by CpG ODN in a syngeneic ALL setting. CpG ODN did not exhibit direct toxicity against cell lines derived from leukemic Eμ-ret transgenic mice in vitro, nor did it alter CD40 or CD86 expression or cytokine production. However, using a flow cytometry-based in vitro killing assay we observed CpG ODN-induced elimination of leukemia cells when cultured with splenocytes or bone marrow cells from Eμ-ret transgene-negative mice (P=0.0388). The difference between CpG ODN-treated and untreated controls became more pronounced with increasing effector:target ratios (P<0.0001). Preliminary data show that depletion of NK cells markedly decreases the magnitude of the observed effect, supporting the hypothesis that this cell type is involved in targeted control of ALL in this model. The ability of CpG ODN to exert anti-leukemia activity in a syngeneic setting suggests that it may have utility as an adjuvant therapy. To test this hypothesis we administered CpG ODN (or PBS) to syngeneic leukemia-bearing mice 2 days after completion of a chemotherapy regimen used to reduce leukemia burden. When mice were sacrificed 3 weeks after treatment, we found significantly reduced leukemia burden in bone marrow (P=0.0019), spleen (P<0.00001) and blood (P=0.00028) of CpG ODN-treated mice. Cell-depletion and cytokine-neutralization assays are currently ongoing to define the mechanism of action of CpG ODN in these settings. To our knowledge, this is the first demonstration of CpG ODN-induced anti-ALL activity in a post-chemotherapy syngeneic model, suggesting that this agent has the potential to treat minimal residual disease and to reduce the incidence of relapse.

Glucocorticoid Induced Cell Death Is Mediated by Its Effect on Cellular Metabolism and Is Independent of Caspase Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3475-3475
Author(s):  
Sandeep Gurbuxani
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Lactate Concentration ◽  
Cellular Metabolism ◽  
Mechanisms Of Resistance ◽  
Metabolic State ◽  
Childhood All ◽  
Mitochondrial Mass

Glucocorticoids (GCs) such as prednisone and dexamethasone are a crucial component of acute lymphoblastic leukemia (ALL) therapy protocols. Multiple studies in childhood ALL have demonstrated that resistance to GC mediated cell death in vitro and in vivo is the single most important predictor of treatment outcome in childhood ALL. However, the mechanisms of GC mediated cell death as well as the mechanisms of resistance are poorly understood. The present study was undertaken to better understand the mechanism of GC induced cell death and to delineate possible mechanisms of resistance. In the initial experiments performed, multiple ALL cell lines, when treated with dexamethasone, underwent a reduction in the amount of reactive oxygen species (ROS) followed by cell cycle arrest and finally cell death which was unaffected by the presence of a pan-caspase inhibitor z-VAD-fmk. Since the amount of ROS present in a cell is an indicator of the metabolic state of the cell, specifically the amount of oxidative phosphorylation going on in the mitochondria, additional experiments were performed to directly estimate the mitochondrial mass as well as the metabolic state of the cells treated with GCs. While the mitochondrial mass measured by Mitotracker green labeling of mitochondria in the viable cells remained unchanged in cell lines susceptible to low concentrations (nano or micromolar) of dexamethasone, there was a prominent reduction in mitochondrial mass 36 hours after dexamethasone exposure in MOLT-4 cell line that requires several fold higher (millimolar) concentration of dexamethasone to induce cell death. The reduction in ROS was not accompanied by an increase in glycolysis as determined by the measurement of lactate concentration in the culture supernatants either in the susceptible or the resistant cells. Since one possible mechanism of reduction in ROS is increased scavenging by molecules that are dependent on the presence of NADPH generated during glucose metabolism via the pentose phosphate pathway (PPP), additional experiments were performed to determine if chemical inhibition of this pathway could augment dexamethasone induced cell death in ALL cell lines. Indeed, addition of transandosterone, an inhibitor of G6PD, the rate limiting enzyme of the PPP, resulted in significantly increased dexamethasone toxicity. Based on these experiments it can be concluded that GC induced cell death is mediated by its effect on cellular metabolism. Furthermore, this cell death is caspase independent and likely proceeds via a pathway mechanistically distinct from classical apoptosis. Finally, cells resistant to GC induced cell death have evolved mechanisms to adapt to GC induced changes in cellular metabolism and may maintain energy production via alternative pathways such as the PPP shunt that are independent of mitochondria.

Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1506-1506
Author(s):  
Marika Masselli ◽  
Serena Pillozzi ◽  
Massimo D'Amico ◽  
Luca Gasparoli ◽  
Olivia Crociani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Map Kinases ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Significant Proportion ◽  
Leukemic Cells ◽  
K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

Therapeutic Targeting of the Bcl2 Family

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-42-SCI-42
Author(s):  
Anthony Letai ◽  
Matthew S. Davids ◽  
Triona Ni Chonghaile ◽  
Jing Deng ◽  
Luv Patel
Keyword(s):  
Cell Death ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
Mitochondrial Outer Membrane ◽  
Short Term ◽  
Provocative Test ◽  
Mitochondrial Outer Membrane Permeabilization

Abstract Many, perhaps most, cancer chemotherapy agents kill cancer cells via the mitochondrial pathway of apoptosis that is controlled by the Bcl-2 family of proteins. Bcl-2 family proteins regulate commitment to cell death by controlling mitochondrial outer membrane permeabilization (MOMP). To better understand how cancer cells commit to apoptosis, and what drugs might make them commit to apoptosis, we have studied perturbing mitochondria with BH3 peptides that are derived from pro-death Bcl-2 family proteins. Using this provocative test, which we call BH3 profiling, we are able to measure how close a cell is to the threshold of apoptosis, a property we call “priming”. Priming corresponds to sensitivity to chemotherapy. Moreover, BH3 profiling can be used to detect dependence on Bcl-2 and Bcl-xL for survival, which predicts cytotoxic response to small molecule antagonists such as ABT-199 and ABT-263. In acute lymphoblastic leukemia, we find that dependence on either Bcl-2 or Bcl-xL varies from case to case, with very important consequences for sensitivity to ABT-199 and ABT-263. In chronic lymphocytic leukemia, ABT-199 has already demonstrated significant clinical activity that corresponds to its on-target activity in mitochondria in vitro. We have been testing how this in vitro mitochondrial activity in BH3 profiling assays might be translated into a useful clinical predictive biomarker. Finally, we can measure how short term incubation with many kinds of drugs, including targeted pathway inhibitors, can increase cancer cell priming, including for primary lymphoid malignancy cells. This short term increase in priming predicts subsequent cancer cell death, including in clinical treatment. We call this method “Dynamic BH3 Profiling” and are exploring how it might best be utilized in the clinic. Disclosures: Letai: Dana-Farber Cancer Institute: Patents & Royalties; AbbVie: Consultancy.

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