scholarly journals GX15-070 (obatoclax) overcomes glucocorticoid resistance in acute lymphoblastic leukemia through induction of apoptosis and autophagy

2010 ◽  
Vol 1 (9) ◽  
pp. e76-e76 ◽  
Author(s):  
N Heidari ◽  
M A Hicks ◽  
H Harada
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Glucocorticoid Resistance ◽  
Induction Of Apoptosis

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.

Obatoclax (GX15-070) Overcomes Glucocorticoid-Resistance In Acute Lymphoblastic Leukemia through Induction of Apoptosis and Autophagy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1809-1809
Author(s):  
Hisashi Harada ◽  
Nastaran Heidari ◽  
Mark Hicks
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Small Molecule ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Single Agent ◽  
Annexin V ◽  
Glucocorticoid Resistance ◽  
Down Regulation

Abstract Abstract 1809 Glucocorticoids (GC) are common components in many chemotherapeutic protocols for lymphoid/myeloid malignancies, including acute lymphoblastic leukemia (ALL). However, patients often develop resistance to GC on relapse. Resistance to GC in ALL can be associated with defects in apoptosis machinery, but not in the GC receptor. Thus, targeting downstream molecules may lead to the development of new therapeutic strategies. GC-induced apoptosis is through the intrinsic mitochondria-dependent pathway. The BCL-2 family proteins are central regulatory proteins in this pathway. We hypothesized that targeting anti-apoptotic MCL-1 might be effective among the BCL-2 family proteins, since (1) we recognized that treatment with dexamethasone (Dex) in CCRF-CEM or Molt-4 T-ALL cells slightly induce MCL-1 and the expression level of MCL-1 is higher in Dex-resistant ALL cells compared with that in Dex-sensitive cells; (2) recent studies have demonstrated that increased expression of MCL-1 associates with GC resistance. In support of our hypothesis, down-regulation of MCL-1 by shRNA enhances Dex-induced cell death. We then pharmacologically inactivate MCL-1 function by GX15-070 (obatoclax), a BH3 mimetic small molecule that targets anti-apoptotic BCL-2 family proteins including BCL-2, BCL-XL, and MCL-1. Treatment with GX15-070 in both Dex-sensitive and -resistant ALL cells shows effective growth inhibition and cell death. GX15-070 induces caspase-3 cleavage and increases Annexin V-positive population, indicative of apoptosis. Before the onset of apoptosis, GX15-070 induces LC3 conversion as well as p62 degradation, both of which are autophagic cell death markers. A pro-apoptotic molecule BAK is released from BAK/MCL-1 complex following GX15-070 treatment. Consistently, down-regulation of BAK reduces caspase-3 cleavage and cell death, but does not alter LC3 conversion. In contrast, down-regulation of ATG5, an autophagy regulator, decreases LC3 conversion and cell death, but does not alter caspase-3 cleavage, suggesting that apoptosis and autophagy induced by GX15-070 are independently regulated. Down-regulation of Beclin-1, which is capable of crosstalk between apoptosis and autophagy, affects GX15-070-induced cell death through apoptosis but not autophagy. Taken together, GX15-070 treatment in ALL could be an alternative regimen to overcome glucocorticoid resistance by inducing BAK-dependent apoptosis and ATG5-dependent autophagy. Enhanced anti-apoptotic BCL-2 family protein expression has been observed in several types of tumors. Targeting these proteins is therefore an attractive strategy for restoring the apoptosis process in tumor cells. Among the small molecule BCL-2 inhibitors, ABT-737 and its analog ABT-263 are the leading compounds currently in clinical development. However, these molecules have an affinity only with BCL-2 and BCL-XL, but not with MCL-1. Thus, ABT-737 can not be effective as a single agent therapeutic for ALL when MCL-1 is overexpressed. In contrast, GX15-070 can overcome the resistance conferred by high level of MCL-1. Our results suggest that GX15-070 could be useful as a single agent therapeutic against ALL and that the activity/expression of anti-apoptotic proteins could be a biomarker to determine the treatment strategy to ALL patients. (Supported by NIH R01CA134473 and the William Lawrence and Blanche Hughes Foundation) Disclosures: No relevant conflicts of interest to declare.

The Wee1 Inhibitor, MK-1775, Sensitizes Leukemic Cells to Different Antineoplastic Drugs Interfering with DNA Damage Response Pathway

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1276-1276 ◽  
Author(s):  
Andrea Ghelli Luserna Di Rora ◽  
Ilaria Iacobucci ◽  
Neil Beeharry ◽  
Simona Soverini ◽  
Cristina Papayannidis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Viability ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Additive Effect ◽  
Proliferation Index ◽  
Induction Of Apoptosis

Abstract Due to inadequate treatments, the survival rate of adult Acute Lymphoblastic Leukemia (ALL) patients with the exclusion of patients with particular genetic alterations, like the Philadelphia positive patients, is still very low. Moreover even the rate of patient that responds to specific treatment develops relapses during their life. Thus there is a need to improve the efficacy of conventional therapy and to discover novel specific targets. In eukaryotic cells Wee1, ATR/Chk1 and ATM/Chk2 are three pathways involved in cell cycle regulation, DNA damages response and DNA repair. Wee1 is a checkpoint kinase, involved mainly in the regulation of G2/M transition through the inhibitory phosphorylation of both Cyclin-dependent kinase 1 (CDK1) and 2 (CDK2) respectively. This study evaluates the effectiveness of MK-1775, a selective Wee1 inhibitor, as a monotherapy and as chemosensitizer agent for the treatment of B-/T-Acute Lymphoblastic Leukemia. Human B (BV-173, SUPB-15, NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines were tested in this study. MK-1775 alone strongly reduced the cell viability in a dose and time-dependent manner in all the cell lines treated. The anti-proliferative activity of MK-1775 was accompanied by an increase in apoptotic cells (AnnexinV/Pi staining) and by DNA damage markers (gH2AX and Parp-1 cleavage). Moreover the inhibition of Wee1 disrupted the cell cycle profile by arresting the cells in late S and in G2/M phase. We hypothesized that targeting Chk1, a kinase upstream, of Wee1, would be more effective in reducing cell proliferation. Indeed, the concomitant inhibition of Chk1 and Wee1 kinases, using the PF-0477736 in combination with MK-1775, synergized in the reduction of the cell viability, inhibition of the proliferation index and induction of apoptosis. Moreover the immunofluorescence staining for the DNA damage marker gH2AX and the mitotic marker phosphor-Histone H3 showed that co-treatment with MK-1775 and PF-0477736 induced cell death by mitotic catastrophe. We undertook further studies to understand the immediate clinical potential of the compound, thus MK-1775 was combined with different drugs (Clofarabine, Bosutinib Authentic, and a particular isomer of this compound).The combination between MK-1775 and clofarabine showed an additive effect in terms of reduction of the cell viability and induction of apoptosis. Finally the Wee1 inhibitor was combined with the tyrosine kinase inhibitors Bosutinib and Bos-isomer (Bos-I). Both the isomers in combination with MK-1775 showed an additive effect in term of reduction of the cell viability. Interestedly the cytotoxic effect of Bos-I was stronger on the Philadelphia-negative cell lines in comparison to the positive counterpart. Western blot analysis highlighted that this compound, but not the Bosutinib authentic, interfered with the Chk1/Chk2 and Wee1 pathway. This supported our previous studies showing that Bosutinib and its isomer possess off-target effects against both Wee1 and Chk1 kinases and thus maybe used as a chemosensitizer (Beeharry et al. Cell Cycle 2014). The results of this study in our opinion identify the Wee1 kinase as a promising target for the treatment of ALL not only as a monotherapy but also as chemosensitizer agent to increase the cytotoxicity of different kind of drugs already used in clinical trials. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; AMGEN: Consultancy; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy.

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