scholarly journals Targeting Mutant p53 in Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 903-903
Author(s):  
Salih Demir ◽  
Galina Selivanova ◽  
Eugen Tausch ◽  
Lisa Wiesmüller ◽  
Stephan Stilgenbauer ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Cell Lines ◽  
Therapeutic Intervention ◽  
High Performance ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Therapy Resistance ◽  
Leukemia Cells

Abstract Mutations of the tumor suppressor gene TP53 have been described to be associated with aggressive disease and inferior prognosis in different types of cancer, including hematological malignancies. In acute lymphoblastic leukemia (ALL), TP53 alterations are infrequently found at diagnosis but have recently been described in about 12% of patients at relapse. This suggests an association with therapy resistance in high risk/relapsed ALL and patients with TP53 mutated ALL have in fact an inferior outcome. Small molecule compounds targeting mutated TP53 such as APR-246, initially described as PRIMA-1MET (p53-dependent reactivation and induction of massive apoptosis) leading to apoptosis induction have shown activity in several types of malignancies with mutated TP53. In ALL, however, mutant TP53 has so far not been addressed as a target for therapeutic intervention. In this study, we investigated a large cohort of patient-derived pediatric B cell precursor (BCP)-ALL primograft samples to identify cases with mutated TP53. Further, we analyzed the effects of APR-246 and evaluated its activity on BCP-ALL cell lines and primografts with mutated (mut) orwild type (wt) TP53. Altogether, 62 BCP-ALL primograft samples established from patients at diagnosis (n=53) or relapse (n=9) by transplantation of primary ALL cells onto NOD/SCID mice were screened for TP53 mutations by denaturating high-performance liquid chromatography (dHPLC) followed by Sanger sequencing of exons 4 to 10 to confirm detected mutations. We identified 4 cases with TP53 mut, 3 obtained from diagnosis (5.6%) and one at relapse (11.1%), corresponding to frequencies described in clinical studies. Mutated cases were further analyzed by fluorescence in situ hybridization (FISH), revealing a 17p deletion in one TP53 mut sample. Similarly, we analyzed 6 BCP-ALL cell lines and identified 2 TP53 mut and 4 TP53 wt lines. Exposure of BCP-ALL primograft (TP53 mut n=4, TP53 wt n=4) and cell line (TP53 mut n=2, TP53 wt n=4) samples to the DNA damaging agent doxorubicin showed, as expected, resistance of TP53 mut leukemia cells for cell death induction, reflected by significantly higher half maximal inhibitory concentrations (IC50; TP53 mut 49 and 143 ng/ml, TP53 wt mean 12 ng/ml) and lower induction of cell death (TP53 mut 16 to 23%, TP53 wt 10 to 60%) in TP53 mut ALL, corroborating the tumor-suppressive function of p53 in ALL. We then investigated the sensitivity of BCP-ALL cell lines for cell death induction by APR-246 (kindly provided by Aprea, Stockholm, Sweden). We observed high sensitivity for APR-246 in TP53 mut (IC50: 5 µM for both cell lines) as compared to TP53 wt ALL (mean IC50: 58 µM). DNA fragmentation and Annexin-V/propidium-iodide (PI) positivity revealed apoptosis as mechanism of APR-246 mediated cell death. Reactive oxygen species (ROS) have recently been described to mediate APR-246 induced cell death in multiple myeloma cells. Therefore, we investigated ROS levels by detection of oxidation-specific fluorescence of dichlorodihydrofluorescein diacetate (DCFDA) in ALL cells. Interestingly, ROS quenching by N-acetyl cysteine abolished induction of cell death in TP53 mut but not TP53 wt ALL cells indicating ROS as a mediator of APR-246 induced cell death in TP53 mut ALL. Furthermore, we addressed p53 activation in response to APR-246 by assessing phosphorylation of p53 (p53pSer15) using phosphoflow cytometry. Most interestingly, APR-246 led to 6-fold increased p53pSer15 levels in TP53 mut compared to no activation in TP53 wt leukemia cells, indicating restoration of p53function upon APR-246treatment in BCP-ALL. Based on these findings, we addressed the effectivity of APR-246on primary, patient-derived primografts and compared sensitivities for cell death induction in TP53 mut (n=4) and TP53 wt (n=4) samples. Importantly, the pattern of responsiveness of TP53 mut ALL was also identified in TP53 mut patient-derived ALL samples with induction of significantly higher cell death rates in TP53 mut ALL (TP53 mut 48%, TP53 wt 18%, 5 µM APR-246, 24 h). Taken together, we showed that TP53 mut BCP-ALL can be targeted by APR-246 leading to re-activation of p53, induction of ROS dependent apoptosis and effective leukemia cell killing. Thus, targeting and re-activation of mutated p53 provides a promising novel strategy for therapeutic intervention in this high-risk subtype of BCP-ALL. Disclosures Selivanova: Aprea: Patents & Royalties: APR-246. Tausch:Gilead: Other: Travel support. Stilgenbauer:Gilead: Honoraria, Research Funding.

Preclinical Evaluation of Oncolytic Reovirus in Targeted Therapeutics for High-Risk Pediatric Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3571-3571
Author(s):  
Matthew F. Clarkson ◽  
Aru Narendran ◽  
Randal N. Johnston
Keyword(s):  
Cell Death ◽  
High Risk ◽  
Cell Viability ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Treatment Strategies ◽  
Leukemia Cells ◽  
Western Blots ◽  
Pediatric Leukemia ◽  
Leukemia Cell Lines

Abstract Abstract 3571 Purpose: Leukemia is the most common malignancy in children. Improved treatment strategies in recent decades have yielded substantially enhanced outcomes for children with leukemia, reaching survival rates >80%. However, there remain significant issues with current treatment. Certain subgroups of patients who are resistant to or relapse from current treatments have a dismal prognosis. Furthermore, there are significant late effects of intensive treatments, including secondary cancers, neurocognitive defects, cardiotoxicity, obesity and infertility. For these reasons, novel treatment strategies are urgently needed for high-risk leukemia in children. Reovirus type 3 Dearing is a wild-type double-stranded RNA virus that has shown great promise as a selective oncolytic agent by its ability to replicate in transformed cells but not in normal cells. Although a number of early phase clinical studies have been completed in patients with advanced, refractory solid tumors in adults, systematic evaluation of this agent in the treatment of refractory pediatric leukemia has not been reported. As an initial step towards developing an oncolytics based treatment approach, we report preclinical data with respect to the activity, target validation, target modulation and drug combinability of reovirus in childhood leukemia cells. Experimental Design: A panel of pediatric leukemia cell lines representing high-risk molecular features such as Bcr-Abl, MLL rearranged and mixed lineage was used (n =6). Expression of JAM-A, the cell surface receptor for reovirus, was assessed by flow cytometry. The Ras Activation Assay Kit (EMD Millipore) was used to assess activity of the RAS protein. Western Blots were used to assess the activation (phosphorylation) of the signaling partners downstream of RAS. Cells treated with reovirus, chemotherapy drugs, or both for distinct treatment schedules were assessed for cell viability by the CellTiter-Glo© Luminescent Cell Viability Assay (Promega), and cell death by apoptosis was confirmed by cleavage of PARP. Productive viral infection was assessed by measuring reoviral protein synthesis by Western Blots, and reoviral replication was assessed by virus plaque titration assay. Drug synergies were calculated according to the method of Chou and Talalay. Results: Target validation assays showed the expression of JAM-A, which facilitates effective viral entry into malignant cells, in five of six cell lines. These cell lines also demonstrated differential activation of RAS and downstream kinases, suggesting targeted susceptibility of these cells to reovirus oncolysis. To further test this, we infected cells with reovirus for 1–4 days and assessed cytopathic effects. Using phase contrast microscopy, we observed the virus treated cell lines to demonstrate morphological changes characteristic of cell death following infection. Cell viability assays were used to quantify this effect, and the mechanism of cell death was determined to be apoptotic as evidenced by caspase-dependent cleavage of PARP. Reovirus-induced cell death was correlated with viral protein production and replication. Next, we screened for the ability of reovirus to induce synergistic activity in a panel of conventional and novel targeted therapeutic agents. Our studies showed that, in contrast to the current antileukemic agents, the Bcl-2 inhibitor BH3 mimetic ABT-737 was able to significantly synergize with reovirus in all cell lines tested. Conclusions: In our in vitro studies, oncolytic reovirus as a single agent showed potent oncolytic activity against all pediatric leukemia cell lines tested that express the receptor for reovirus, regardless of the status of the RAS signaling pathway. Further, we found reovirus-induced oncolysis can be enhanced by combination with Bcl-2 inhibition but was unaltered or antagonized by the other drugs indicating a key relationship between the two pathways. As such, our data for the first time, show that pediatric leukemia cells carry the potential to be targeted by reovirus induced oncolysis and the identification of drug synergy and the biomarkers of target modulation provide the basis for further studies to develop this novel therapeutic approach for clinical studies in the near future. Disclosures: No relevant conflicts of interest to declare.

Gene Delivery to Human B-Precursor Acute Lymphoblastic Leukemia Cells

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3537-3545 ◽  
Author(s):  
Leo Mascarenhas ◽  
Renata Stripecke ◽  
Scott S. Case ◽  
Dakun Xu ◽  
Kenneth I. Weinberg ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Gene Transfer ◽  
Gene Delivery ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Virus ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Reh Cells ◽  
Hiv 1

Autologous leukemia cells engineered to express immune-stimulating molecules may be used to elicit antileukemia immune responses. Gene delivery to human B-precursor acute lymphoblastic leukemia (ALL) cells was investigated using the enhanced green fluorescent protein (EGFP) as a reporter gene, measured by flow cytometry. Transfection of the Nalm-6 and Reh B-precursor ALL leukemia cell lines with an expression plasmid was investigated using lipofection, electroporation, and a polycationic compound. Only the liposomal compound Cellfectin showed significant gene transfer (3.9% to 12% for Nalm-6 cells and 3.1% to 5% for Reh cells). Transduction with gibbon-ape leukemia virus pseudotyped Moloney murine leukemia virus (MoMuLV)-based retrovirus vectors was investigated in various settings. Cocultivation of ALL cell lines with packaging cell lines showed the highest transduction efficiency for retroviral gene transfer (40.1% to 87.5% for Nalm-6 cells and 0.3% to 9% for Reh cells), followed by transduction with viral supernatant on the recombinant fibronectin fragment CH-296 (13% to 35.5% for Nalm-6 cells and 0.4% to 6% Reh cells), transduction on human bone marrow stroma monolayers (3.2% to 13.3% for Nalm-6 cells and 0% to 0.2% Reh cells), and in suspension with protamine sulfate (0.7% to 3.1% for Nalm-6 cells and 0% for Reh cells). Transduction of both Nalm-6 and Reh cells with human immunodeficiency virus–type 1 (HIV-1)–based lentiviral vectors pseudotyped with the vesicular stomatitis virus-G envelope produced the best gene transfer efficiency, transducing greater than 90% of both cell lines. Gene delivery into primary human B-precursor ALL cells from patients was then investigated using MoMuLV-based retrovirus vectors and HIV-1–based lentivirus vectors. Both vectors transduced the primary B-precursor ALL cells with high efficiencies. These studies may be applied for investigating gene delivery into primary human B-precursor ALL cells to be used for immunotherapy.

scholarly journals Dynamin inhibition causes context-dependent cell death of leukemia and lymphoma cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0256708
Author(s):  
Christopher von Beek ◽  
Linnéa Alriksson ◽  
Josefine Palle ◽  
Ann-Marie Gustafson ◽  
Mirjana Grujic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Tumor Formation ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Bone Marrow Biopsies

Current chemotherapy for treatment of pediatric acute leukemia, although generally successful, is still a matter of concern due to treatment resistance, relapses and life-long side effects for a subset of patients. Inhibition of dynamin, a GTPase involved in clathrin-mediated endocytosis and regulation of the cell cycle, has been proposed as a potential anti-cancer regimen, but the effects of dynamin inhibition on leukemia cells has not been extensively addressed. Here we adopted single cell and whole-population analysis by flow cytometry and live imaging, to assess the effect of dynamin inhibition (Dynasore, Dyngo-4a, MitMAB) on pediatric acute leukemia cell lines (CCRF-CEM and THP-1), human bone marrow biopsies from patients diagnosed with acute lymphoblastic leukemia (ALL), as well as in a model of lymphoma (EL4)-induced tumor growth in mice. All inhibitors suppressed proliferation and induced pronounced caspase-dependent apoptotic cell death in CCRF-CEM and THP-1 cell lines. However, the inhibitors showed no effect on bone marrow biopsies, and did not prevent EL4-induced tumor formation in mice. We conclude that dynamin inhibition affects highly proliferating human leukemia cells. These findings form a basis for evaluation of the potential, and constraints, of employing dynamin inhibition in treatment strategies against leukemia and other malignancies.

Single-Cell Phospho-Profiling in Pediatric Acute Lymphoblastic Leukemia (ALL) Reveals Constitutive and Cytokine Induced Specific Signatures Involving Stat5

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2511-2511
Author(s):  
Manon Queudeville ◽  
Hannah Kunze ◽  
Sarah M. Eckhoff ◽  
Klaus-Michael Debatin ◽  
Lueder H. Meyer
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Single Cell ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Leukemia Cell Lines ◽  
Significant Difference

Abstract Oncogenesis and tumor progression are supported by alterations in cellular signaling. We used phospho-specific antibodies in flow cytometry to analyze specific signaling profiles of leukemia cells at a single cell level in 7 B cell precursor (BCP)-ALL leukemia cell lines and 7 primary pediatric BCP-ALL xenograft samples. Peripheral blood lymphocytes gated on CD19-positive B cells were used as normal nonmalignant controls. Cells were stimulated by different stimulants and cytokines (PMA, anisomycin, IL-4, IL-6, IL-7, IL-10 and IFN-α) and activation of various phosphoepitopes (pERK, pp38, pJNK, pStat1, pStat3, pStat5, pStat6) was analyzed and compared to the basal state of unstimulated samples. Signaling profiles of normal B-lymphocytes were compared to those of the BCPALL cell lines as well as to the BCP-ALL xenograft samples. Significance of differences was assessed by the nonparametric Mann-Whitney U-test. Basal phosphorylation was significantly higher in the leukemia cell lines than in normal lymphocytes. Similarly, basal phosphorylation of all analyzed epitopes in xenografts exceeded the phosphorylation state of normal B-lymphocytes (with the exception of p38 phosphorylation, where there was no significant difference). Interestingly, the BCP-leukemia cell lines also had significantly higher basal phosphorylation levels than the primary BCP-ALL xenografts. However, when comparing the amounts of phosphorylation before and after stimulation mature normal B-cells displayed significantly higher profiles compared to the leukemia cell lines e.g. for pp38 and pJNK after stimulation with PMA (P= .001), for pStat3 after stimulation with IL-6 (P= .002) and IL-10 (P= .037) and for pStat6 (P= .001) after stimulation with IL-4. Conversely, the leukemia cell lines showed increased phosphorylation of p38 after stimulation with anisomycin (P= .021) as well as higher Stat5 phosphorylation after stimulation with IL-7 (P= .021) compared to normal lymphocytes. In normal B-cells compared to xenografts higher levels were found after stimulation with PMA for pp38 (P= .007), for pJNK after PMA stimulation (P= .001), for pStat3 after IL-6 (P=.003) and for pStat6 after IL-4 (P= .002) stimulation while the xenograft samples displayed stronger reaction to stimulation with anisomycin for pp38 (P= .037) and to stimulation with IL-7 for pStat5 (P= .028). The level of phosphorylation after treatment with different stimulants in the xenografted leukemia samples was similar to that of the leukemia cell lines although the cell lines displayed higher basal phosphorylation values. The BCP-leukemia cell lines and the BCP xenograft samples both displayed high levels of constitutive phosphorylation in general reducing their ability to react to a given stimulus compared to normal B-lymphocytes. With the most important exception of Stat5: we consistently found that Stat5 phosphorylation is increased in acute lymphoblastic leukemia cell lines and primary xenografts after stimulation with IL-7 compared to normal B-lymphocytes. Stat5 is known to enhance proliferation and protect from apoptosis and our data now strongly suggest that Stat5 and Stat5 dependent pathways are critically involved in leukemogenesis. Since we could identify significant and specific phosphorylation signatures characteristic for leukemia cells, this provides a strategy to define pathways important for continued survival, proliferation and resistance of leukemia and allows identification of therapeutic targets and novel biomarkers associated with clinical outcome.

Diverse pathways mediate chemotherapy-induced cell death in acute lymphoblastic leukemia cell lines

APOPTOSIS ◽  
2006 ◽  
Vol 11 (11) ◽  
pp. 1977-1986 ◽  
Author(s):  
Dong-Joon Min ◽  
Naomi P. Moskowitz ◽  
Carrie Brownstein ◽  
Hokyung Lee ◽  
Terzah M. Horton ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cell Lines

scholarly journals Synergistic Activity of the MCL-1-Specific Inhibitor S63845 with Venetoclax in B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1416-1416
Author(s):  
Felix Seyfried ◽  
Felix Stirnweiß ◽  
Stefan Köhrer ◽  
Klaus-Michael Debatin ◽  
Lüder Hinrich Meyer
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Specific Inhibitor ◽  
Synergistic Activity ◽  
Cell Precursor

Abstract Deregulated cell death and survival pathways contribute to leukemogenesis and treatment failure of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. The intrinsic apoptosis pathway is regulated at the mitochondrial level by different pro- and anti-apoptotic molecules. Members of the BCL-2 family are key regulators of mitochondrial apoptosis signaling. Pro-apoptotic BH3-only proteins like BIM and BID activate pro-death proteins such as BAX and BAK leading to cell death. Anti-apoptotic BCL-2 family members including BCL-2, BCL-XL and MCL-1 bind to and sequester pro-apoptotic molecules, prevent activation of pro-death proteins and counter-regulate apoptosis induction. Small molecule inhibitors have been developed that block binding to anti-apoptotic molecules like BCL-2, leading to release of pro-apoptotic proteins and cell death induction. In particular, the BCL-2-specific inhibitor venetoclax (VEN) has demonstrated substantial anti-cancer activity and became an approved drug for the treatment of CLL patients. Investigating different, individual BCP-ALL samples, we and others recently identified heterogeneous sensitivities for VEN, suggesting that BCP-ALL cells might also depend on other pro-survival BCL-2 family proteins including MCL-1, leading to VEN insensitivity and resistance. A novel BH3-mimetic, S63845, that selectively targets MCL-1 has been reported. Here, we assessed the activity of S63845 and addressed a potential synergism of simultaneous blockage of BCL-2 and MCL-1 by VEN and S63845 (S) in BCP-ALL. The activity of the MCL-1 inhibitor was analyzed in a panel of BCP-ALL cell lines (N=6) and a series of primary, patient-derived BCP-ALL primograft samples (N=27) determining half-maximal effective concentrations (EC50) upon exposure to increasing concentrations of S and analysis of cell death induction. We observed heterogeneous sensitivities to S with EC50 values ranging from 16 nM to almost 10 µM. Protein expression of MCL-1 and other BCL-2 family members BCL-2, BCL-XL and BCL-W was assessed by western blot analysis and quantified, however neither association of MCL-1 levels nor expression of the other regulators and S sensitivity was found in cell lines and primograft leukemias. Moreover, we also compared sensitivities for both inhibitors but found independent activities of S and VEN in individual ALL samples. Next, we addressed the role of MCL-1 for VEN sensitivity and generated two MCL-1 knock out BCP-ALL cell lines by CRISPR/Cas9 gene editing. In both lines, clearly increased VEN sensitivities were observed upon depletion of MCL-1, indicating that MCL-1 is contributing to activity of the BCL-2 inhibitor VEN. Based on these findings, we investigated the effects of pharmacological MCL-1 inhibition for VEN sensitivity and incubated all 6 cell lines with VEN and S at increasing concentrations and observed clear synergistic effects upon combined BCL-2 and MCL-1 inhibition indicated by combination indices (CI) below 0.1. Moreover, we investigated 7 primograft BCP-ALL samples and found that MCL-1 inhibition by S clearly synergized with VEN activity (CI < 0.3). To investigate the anti-leukemia activity of co-targeting BCL-2 and MCL-1 in vivo in a pre-clinical setting, a high-risk leukemia derived from an infant, MLL/ENL rearranged pro-B ALL case was transplanted onto NOD/SCID mice. Upon ALL manifestation (presence of >5% human blasts in blood), recipients were treated with either VEN, S, the combination of both, or vehicle for 10 days. After treatment, leukemia loads were analyzed showing significantly reduced loads in the co-treated group as compared to vehicle, VEN or S alone in spleen, bone marrow, and central nervous system (p-values < 0.05), indicating synergistic activity of co-inhibition of BCL-2 and MCL-1 in vivo. Taken together, our data show heterogeneous sensitivity of individual BCP-ALL samples to MCL-1 inhibition by S, which is not associated with MCL-1 protein expression levels or VEN sensitivity. Both, genetic depletion and inhibition of MCL-1 by S synergizes with VEN leading to increased anti-leukemia activity in vitro and ex vivo. Importantly, co-targeting BCL-2 and MCL-1 significantly reduced leukemia infiltration in spleen, BM and CNS in a pre-clinical model of high-risk BCP-ALL, warranting further evaluation and possible clinical application of targeting MCL-1 alone and in combination with BCL-2 inhibition. Disclosures No relevant conflicts of interest to declare.

JAZ “Targeting” Compounds Induce G1 Cell Cycle Arrest Followed by Cell Death in Human Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2658-2658
Author(s):  
Mingli Yang ◽  
George Q. Yang ◽  
Jinghua Jia ◽  
David Ostrov ◽  
W. Stratford May
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Cycle Arrest ◽  
G1 Cell Cycle Arrest

Abstract JAZ (just another zinc finger protein) was previously identified in our laboratory as a unique ZFP that preferentially binds to double-stranded (ds) RNA rather than dsDNA. We found that interleukin-3 growth factor withdrawal upregulates JAZ expression in factor-dependent hematopoietic cells in association with p53 activation and induction of apoptotic cell death. We recently discovered JAZ as a novel direct, positive regulator of p53 transcriptional activity. The mechanism involves direct binding to p53’s C-terminal (negative) regulatory domain to activate “latent” p53 in response to non-genotoxic stress signals. Our preliminary data indicate that JAZ is differentially expressed in murine and human bone marrow cells and in normal and malignant hematopoietic tissues and cell lines. Thus, we have explored JAZ as a potentially novel molecular target in human leukemia by identifying small molecules that bind and activate JAZ. Using a high-throughput, “molecular docking” strategy, we have screened approximately 240,000 small molecules for their ability to interact with JAZ. Based on the Lipinski Rules for Drug Likeness (molecular characteristics favorable for absorption and permeability), we identified ~70 putative “drug-like” binding molecules with high scores and obtained ~40 of them from the NCI Developmental Therapeutics Program. We first tested their cytotoxic effect on various human leukemia cell lines including wt p53 expressing Reh pre-B lymphoblastic leukemia and Molt-3 T-cell lymphoblastic leukemia cells, and p53-deficient U937 leukemic monocyte lymphoma and KU812 and K562 chronic myelogenous leukemia cells. We have selected four “candidate” JAZ-targeting (J1-J4) compounds for further investigation because they are potent (IC50 = &lt;1 to ~50 μM) in killing leukemia cells in association with upregulation of JAZ protein expression and p53 activation. Since we previously demonstrated that JAZ can induce G1 cell cycle arrest prior to apoptosis in NIH3T3 mouse fibrablast cells in association with upregulation of p21, dephosphorylation of Rb and repression of cyclin A, we have tested these J-compounds for their potential effect on cell cycle progression. Drug treatment followed by flow cytometry analysis was carried out in human leukemia cell lines. Results reveal that the J2, J3 and J4 but not J1 compounds induce significant G1 cell cycle arrest followed by cell death in a dose- and time-dependent manner (e.g. an increase in the G1 population by up to 35 % at 24 hr following the treatment at doses of 0.1 to 50 μM). These data indicate that the J2-J4 compounds can not only induce leukemia cell killing but also mediate growth arrest. Interestingly, J3 and J4 are FDA-approved drugs (for the treatment of non-cancer diseases), suggesting a potentially novel role for these clinically available drugs as therapy for hematologic malignancies. Therefore, while further in vitro and in vivo characterization remains to be carried out, the JAZ-“targeting” compound(s) points the way to develop a potentially novel therapeutic strategy targeting JAZ to treat human leukemia.

Gene Delivery to Human B-Precursor Acute Lymphoblastic Leukemia Cells

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3537-3545 ◽  
Author(s):  
Leo Mascarenhas ◽  
Renata Stripecke ◽  
Scott S. Case ◽  
Dakun Xu ◽  
Kenneth I. Weinberg ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Gene Transfer ◽  
Gene Delivery ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Virus ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Reh Cells ◽  
Hiv 1

Abstract Autologous leukemia cells engineered to express immune-stimulating molecules may be used to elicit antileukemia immune responses. Gene delivery to human B-precursor acute lymphoblastic leukemia (ALL) cells was investigated using the enhanced green fluorescent protein (EGFP) as a reporter gene, measured by flow cytometry. Transfection of the Nalm-6 and Reh B-precursor ALL leukemia cell lines with an expression plasmid was investigated using lipofection, electroporation, and a polycationic compound. Only the liposomal compound Cellfectin showed significant gene transfer (3.9% to 12% for Nalm-6 cells and 3.1% to 5% for Reh cells). Transduction with gibbon-ape leukemia virus pseudotyped Moloney murine leukemia virus (MoMuLV)-based retrovirus vectors was investigated in various settings. Cocultivation of ALL cell lines with packaging cell lines showed the highest transduction efficiency for retroviral gene transfer (40.1% to 87.5% for Nalm-6 cells and 0.3% to 9% for Reh cells), followed by transduction with viral supernatant on the recombinant fibronectin fragment CH-296 (13% to 35.5% for Nalm-6 cells and 0.4% to 6% Reh cells), transduction on human bone marrow stroma monolayers (3.2% to 13.3% for Nalm-6 cells and 0% to 0.2% Reh cells), and in suspension with protamine sulfate (0.7% to 3.1% for Nalm-6 cells and 0% for Reh cells). Transduction of both Nalm-6 and Reh cells with human immunodeficiency virus–type 1 (HIV-1)–based lentiviral vectors pseudotyped with the vesicular stomatitis virus-G envelope produced the best gene transfer efficiency, transducing greater than 90% of both cell lines. Gene delivery into primary human B-precursor ALL cells from patients was then investigated using MoMuLV-based retrovirus vectors and HIV-1–based lentivirus vectors. Both vectors transduced the primary B-precursor ALL cells with high efficiencies. These studies may be applied for investigating gene delivery into primary human B-precursor ALL cells to be used for immunotherapy.

scholarly journals FOXO1 Is Involved in the Regulation of B-Cell Precursor Acute Lymphoblastic Leukemia Survival and Serves As a Novel Target for Directed Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4020-4020
Author(s):  
Salih Demir ◽  
Fan Wang ◽  
Franziska Gehringer ◽  
Clarissa Weitzer ◽  
Klaus-Michael Debatin ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Precursor ◽  
Free Survival

Abstract Acute lymphoblastic leukemia (ALL) is the most common pediatric and adolescent malignancy. Although current treatment provides five-year event-free survival, in up to 20% conventional chemotherapy fails resulting in relapse with inferior prognosis. FOXO1 is a member of the forkhead family of transcription factors, which is preferably expressed in B-cells with high expression at the early B-cell stage. FOXOs are involved in several cellular processes including cell death and proliferation, anti-cancer drug resistance and protection from oxidative stress. Since FOXO1 can enhance tumor growth and potentiate metastasis, we aimed to investigate the effects of FOXO1 inactivation on B-cell precursor (BCP)-ALL, including preclinical in vivo evaluation. FOXO1 expression levels were compared among 497 cancer samples using the Genevestigator online software. Expression of FOXO1 in BCP-ALL was significantly higher than in any of the other cancer types. Next, we investigated FOXO1 expression and subcellular localization in 3 BCP-ALL cell lines by cellular fractionation and fluorescent microscopy. Both methods showed localization of FOXO1 in the nucleus, indicating transcriptionally active FOXO1 in BCP-ALL. In order to study the potential anti-tumor effect of FOXO1 repression, we investigated genetically modified, FOXO1 deficient BCP-ALL cell lines (n=5) and observed no cell death induction in control transduced cells, in contrast to a clear reduction of cell viability of up to 80% upon FOXO1 knock-down, clearly indicating dependency of BCP-ALL cells on FOXO1. Moreover, lentiviral mediated FOXO1 knockdown did not induce cell death in the Hodgkin's lymphoma cell line cHL, suggesting a BCP-ALL specific importance for FOXO1. Based on these results indicating the importance of FOXO1 expression for BCP-ALL maintenance, we investigated the feasibility of pharmacological interference with FOXO1. Exposure of 7 BCP-ALL, 4 T-ALL, 3 B-cell NHL, 2 DLBCL and 3 cHL cell lines to the small molecular weight FOXO1 inhibitor AS1842856 showed effectivity in BCP-ALL lines, reflected by significantly higher half maximal inhibitory concentrations (IC50) by MTT test. The most sensitive cell line was the BCP-ALL line RS4;11, while the cHL cell line SUP-HD1 showed insensitivity for FOXO1 inhibition (IC50: 3 nM and 26 µM), again indicating that BCP-ALL is particularly dependent on FOXO1 activity. Caspase 3 cleavage detected upon exposure to AS1842856 showed induction of apoptosis as mechanism of cell death. Furthermore, we evaluated the sensitivity of primary BCP-ALL primograft samples (n=9) exposing the ALL cells to increasing pharmacologically relevant concentrations of AS1842856. The inhibitor increased cell death as measured by flow cytometry (FSC/SSC criteria) in all of the samples tested in a time and dose dependent manner. Importantly, FOXO1 inhibition also showed activity on high risk leukemias including MLL-rearranged and early or second-relapse cases. Moreover, we investigated the in vivo effectivity of AS1842856. Two different patient derived leukemias were transplanted onto NOD/SCID mice and upon leukemia manifestation vehicle or AS1842856 was administered for a time of 11 days. At the end of the experiment, all mice were sacrificed and tumor loads were quantified in spleen, bone marrow and central nervous system (CNS). Importantly, tumor loads of all compartments and spleen sizes were significantly reduced in AS1842856 treated animals (p=0.028, U-test). Moreover, in an early-relapse sample leukemia-free survival upon AS1842856 treatment was evaluated. Mice were treated by vehicle or AS1842856 (n=10/group) during 11 days. Leukemia-free survival was significantly prolonged in mice which received AS1842856 (p=0.003, Log-rank test). Taken together, we show that the active form of FOXO1 is highly expressed in BCP-ALL cells as compared to other cancers, and that viability of BCP-ALL cells is regulated by FOXO1 activity. Importantly, silencing or pharmacological inhibition of FOXO1 induces cell death in BCP-ALL primogafts including high risk cases, both ex vivo and preclinically in vivo. Thus, targeting FOXO1 provides a promising novel strategy for therapeutic intervention in these high-risk subtypes of BCP-ALL. Disclosures No relevant conflicts of interest to declare.

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