scholarly journals Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

10.3791/4442 ◽  
2012 ◽  
Author(s):  
Anna Brestovitsky ◽  
Rakefet Sharf ◽  
Tamar Kleinberger
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Lines

S-Dimethylarsinoglutathione (SGLU/ZIO101) Is a Novel Active Organic Arsenical That Displays a Unique Gene Expression Profile in Myeloma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5043-5043
Author(s):  
Lawrence H. Boise ◽  
Alejo A. Morales ◽  
Claire R. Croutch ◽  
Delia Gutman ◽  
Robert Peter Gale ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Cell Death ◽  
Phase I ◽  
Cell Lines ◽  
Stress Responses ◽  
Cellular Response ◽  
Differential Sensitivity ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract Arsenic Trioxide (ATO) is highly active in acute promyelocytic leukemia (APL) and has activity in several other diseases including multiple myeloma. Since arsenicals are active and it is known that organic arsenicals are less toxic than ATO, the testing of new organic arsenicals is warranted. One such compound, ZIO-101 is in phase I/II studies. Therefore we previously compared the ability of ZIO-101 and ATO to kill four myeloma cell lines (RPMI 8226, U266, KMS11, MM.1s) that display differential sensitivity to ATO. Sensitivity to ATO and ZIO-101 did not correlate, as the most ATO resistant line (RPMI 8226) was highly sensitive to ZIO-101. We and others have reported that glutathione (GSH) is a critical regulator of ATO-induced cell death and we have utilized ascorbic acid (AA) as a GSH depleting agent both in vitro as well as clinically. We therefore also tested the effects of GSH depletion on ZIO-101-induced cell death in MM cell lines. BSO was much more effective at sensitizing cells to ATO than to ZIO-101. Moreover while AA could sensitize cells to ATO, it actually protected cells from cell death induced by ZIO-101. Taken together these data suggest ZIO-101 is active against myeloma cells although factors that determine the potency of this compound are different than those for ATO. To better characterize these differences gene expression profiling of the cellular response to ZIO-101 was performed. RNA was isolated from the 4 cell lines treated with ZIO-101 for 0, 6 and 24 h and profiling performed using Affymetrix Hu133 plus 2 arrays. We initially focused on genes that demonstrated similar changes in all four cell lines. 320 probes demonstrated an increase of 1.5 or greater at 6 h while only 58 increased at 24 h. Additionally 265 genes were decreased by at least 1.5 fold at 6 h while only 12 genes were down regulated 1.5 or greater at 24 h. Interestingly the pattern of gene expression was distinct from that observed in similar experiments with ATO. Most notably genes associated with metal responses (MT-1, ZnT-1) and oxidative stress responses (increased expression of HO-1, NQO-1, malic enzyme, GSH synthesis pathway, ferritin) were either absent or only transiently increased. In contrast there was increased expression of the pro-apoptotic gene Noxa compared to ATO treatment in the 4 cell lines. Taken together these data indicate the cellular response to ZIO-101 does not include the up regulation of protective pathways and suggest that ZIO-101 does not initiate cell death through the induction of oxidative stress. This may reflect differences in either metabolism or mechanism of action. Thus resistance to one form of arsenic does not preclude use of another. A phase I/II study of ZIO-101 in myeloma is underway.

The Functional Impact of PKCβ/PI3K/AKT Signalling on Translational Initiation in MCL

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2624-2624
Author(s):  
Grit Hutter ◽  
Yvonne Zimmermann ◽  
Marc Weinkauf ◽  
Alina Postnikova ◽  
Tobias Weiglein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Lines ◽  
Translation Initiation ◽  
Target Genes ◽  
Mtor Inhibitors ◽  
Potential Effect ◽  
Mtor Pathway ◽  
Akt Pathway ◽  
Mtor Signalling

Abstract Introduction: Mantle cell lymphoma (MCL) is an aggressive form of B-cell non-Hodgkin’s lymphoma (NHL). It is characterized by the t(11;14)(q13;q32) translocation, which results in the overexpression of cyclinD1, a cyclin regulated by the PI3K/AKT pathway. Activation of the PI3K/AKT pathway has been shown to be involved in the pathogenesis of MCL. In addition overexpression of the protein kinase C beta (PKCβ) has been described for most cases of MCL, inhibited by enzastaurin which in turn induces apoptosis and reduces proliferation through the PKCβ/PI3K/AKT pathways. 4EBP1 is described as one of the downstream targets of PI3K/mTOR pathway linking translation initiation with PI3K/mTOR signalling as a EIF4E binding protein and playing therefore critical role in the control of protein synthesis, survival and cell growth. Targeting 4EBP1 and/or EIF4E via the PI3K/AKt/mTOR signalling or directly will affect tumor tissue. Aim of the study: The aim of the study was to determine the functional impact of PKCβ/PI3K/AKt/mTOR signaling on the translation initiation factor EIF4E, its binding protein and regulated proteins in MCL cell lines. Methods: MCL cell lines were treated with inhibitors of the PKCβ/PI3K/AKt/mTOR pathways (enzastaurin, LY294002, rapamycin) for up to 48h.The impact of the drugs on the proliferation rate of the cells was accessed after 48h by WST-assay and/or cell count. mRNA expression levels were determined using Taqmanassays. Protein phosphorylation status and protein expression were identified by westernblot. For downregulation of EIF4E in the cells sodium arsenite was used. Specific silencing of EIF4E was achieved by transfection of cells with siRNA against EIF4E. Results: The MCL celllines (5) responded to the treatment with the inhibitors of the PI3K/AKt/mTOR pathway at a IC50 for rapamycin between 5nM-50nM and for the PI3Kinhibitor between 0,31μM-5μM. Treatment of the cells with the PI3K/AKt/mTOR inhibitors induced dephosphorylation of 4EBP1 in a time-and dosedependent manner while a potential effect of the PI3K and mTOR inhibitors on the EIF4E expression and its target genes (cyclinD1, BCL2) could not be shown consistently. 4 out of 5 MCL cell lines were susceptible to enzastaurin with an IC50 between 2μM-5μM. In the not responding to enzastaurin and most resistant to rapamycin cell line (Rec-1) no 4EBP1 proteinexpression was detectable. Dephosphorylation of 4EBP1 achieved by treatment of the cells with sodium arsenit was accompanied by downregulation of EIF4E, cyclinD1 and BCL2 proteins but also stop of proliferation. The potential involvement of eIF4E gene expression in the NaAsO2-induced cytotoxicity and cell death in MCL cell lines was shown by silencing the expression of the eIF4E gene by transfection with siRNA specifically targeting the eIF4E gene expression leading to downregulation of cyclinD1, 4EBP1 proteins and cell proliferation. Conclusion: Eventhough treatment of the cells with the PI3K/AKt/mTOR inhibitors induced dephosphorylation of 4EBP1 a potential effect of the PI3K and mTOR inhibitors on the EIF4E expression and its target genes (cyclinD1, BCL2) could not be shown consistently. Instead dephosphorylation of 4EBP1 accomponied by downregulation of EIF4E or targeted downregulation of eIF4E gene expression lead to downregulation of cyclinD1 and BCL2 proteins as well as cell death in MCL. Therefore targeting the downstream targets of the PI3K/AKt/mTOR signalling 4EBP1 and/or EIF4E directly seems to be a promising anticancer strategy.

Epigenetic modulation of gene expression of human leukemia cell lines – induction of cell death and senescence

Neoplasma ◽  
2011 ◽  
Vol 58 (1) ◽  
pp. 35-44 ◽  
Author(s):  
K. ELKNEROVA ◽  
D. MYSLIVCOVA ◽  
Z. LACINOVA ◽  
I. MARINOV ◽  
L. UHERKOVA ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Human Leukemia Cell ◽  
Leukemia Cell Lines ◽  
Modulation Of Gene Expression ◽  
Epigenetic Modulation

scholarly journals Gene expression analysis of cell death induction by Taurolidine in different malignant cell lines

BMC Cancer ◽  
2010 ◽  
Vol 10 (1) ◽  
Author(s):  
Ansgar M Chromik ◽  
Stephan A Hahn ◽  
Adrien Daigeler ◽  
Annegret Flier ◽  
Daniel Bulut ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Lines ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Malignant Cell

Insights into the Genetic Signature Associated with Cell Death Induced by an Antibody-Avidin Fusion Protein Specific for Transferrin Receptor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3476-3476
Author(s):  
Gustavo Helguera ◽  
Jose A. Rodriguez ◽  
Tracy R. Daniels ◽  
David Casero Diaz-Cano ◽  
Matteo Pellegrini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
B Cells ◽  
Fusion Protein ◽  
Cell Line ◽  
Cell Lines ◽  
Transferrin Receptor ◽  
Expression Levels ◽  
Genetic Signature ◽  
Buffer Control

Abstract We have previously developed an anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) that exhibits a potent anti-proliferative/pro-apoptotic activity against malignant B cells. This effect has been attributed, at least in part, to the internalization and rapid degradation of the hTfR by anti-hTfR IgG3-Av, resulting in lethal iron deprivation. However, in a panel of eight malignant B cells treated with anti-hTfR IgG3-Av we observed varying levels of sensitivity among the cell lines, where IM-9 (a human lymphoblastoid cell line) showed high sensitivity, while U266 (a human multiple myeloma cell line) was significantly resistant; although both cell lines express high levels of hTfR. To elucidate the mechanisms behind resistance and sensitivity of these human malignant B cells we conducted a global gene expression comparison treating the two cell lines for 0, 1, 3, 9, and 24 hours with anti-hTfR IgG3-Av or buffer control. Samples of total RNA were collected in duplicate at each time point and gene expression levels analyzed using HumanRef-8 v2 Expression BeadChips (Illumina Inc. San Diego, CA). Microarray data were preprocessed using quantile normalization and the difference in levels of gene expression was established using Bayesian statistics. Out of 8,095 array probes detected at time zero in both IM-9 and U266 cell lines, 167 had a greater than four-fold significant difference (regularized bayesian t-test p<0.05). With the genes selected by this global comparison, we performed a gene ontology analysis to identify genes associated with functional processes that may be involved in the differential sensitivity to anti-hTfR IgG3-Av. We found that genes facilitating apoptotic signaling through RAS, MAPK, and P53 (such as PIK3CD and CBLB) were expressed at significantly higher levels in IM-9 compared to U266, while genes involved in cell cycle including the cyclin-dependent growth regulator CDKN2C and a number of core histones were more highly expressed in U266 compared to IM-9. Of all the genes present in both cell lines, a time course study of 24 hour treatment with anti-hTfR IgG3-Av identified 74 genes in IM-9 cells and 10 genes in U266 cells with significant changes (p<0.05) at greater than 1.5-fold different expression levels compared to buffer control treated cells. IM-9 cells treated with anti-hTfR IgG3-Av showed increased expression of a number of apoptosis related genes including GADD45A, IKIP, DDB2, and TP53I3 at 24 hours. In contrast, U266 cells treated with anti-hTfR IgG3-Av showed decreased expression of genes involved in apoptosis induction and cell death such as PLEKHF1, OKL38, and CDC14B. Although further studies are needed to validate the expression changes, these results illustrate the complexity of the mechanism responsible of cell death induced by anti-hTfR IgG3-Av, and suggest that the molecular pathway involving p53 may be associated with cell death in the sensitive cell line. These studies are expected to pave the way to fully elucidate the mechanisms responsible for cell death induced by anti-hTfR IgG3-Av and the identification of a genetic signature responsible for resistance to this therapeutic.

Pan-Anti-Apoptotic BCL-2 Family Inhibitor, Obatoclax, Activates Autophagic Cell Death Pathway and Has Potent Cytotoxicity in Infant and Pediatric MLL-Rearranged Leukemias

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2647-2647
Author(s):  
Alena Y. Zhang ◽  
Blaine W. Robinson ◽  
Li-San Wang ◽  
Kajia Kao ◽  
Lori Cory ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Lines ◽  
Family Members ◽  
Rt Pcr ◽  
Basal Expression ◽  
Cell Death Pathway ◽  
Vehicle Treatment ◽  
Change In Mean ◽  
Mtt Assays

Abstract Cell death pathways are desired targets of small molecule inhibitors since their deregulation plays an important role in chemotherapy resistance. Obatoclax binds to the BH3 pocket of anti-apoptotic BCL-2 family proteins, inhibiting their interactions with pro-apoptotic BCL-2 family members. Before we found potent obatoclax activity in MLL/AF4+ cell lines and 6 MLL rearranged (MLL+) leukemias. In apoptosis assays of the cell lines obatoclax increases TUNEL staining but minimally activates caspase 3 (Rege ASH 2005; Zhang AACR 2007). In this study, we tested the cytotoxicity of obatoclax in additional MLL+ leukemias and investigated its mechanism of action with a focus on autophagy (ATG), since ATG proteins such as beclin 1 also interact with anti-apoptotic BCL-2 family members. Methods: MLL partner genes were determined by molecular/cytogenetic methods. 17 primary MLL+ leukemias including the original 6 (12 ALL/11 infants, 1 child; 3 AML/1 each infant, child, adolescent; 2 bilineal/2 infants) were tested in MTT assays after 72 h obatoclax exposures. MTT assays on cytotoxic drug-obatoclax combinations were performed in a primary MLL/AF4+ ALL and interactions were studied by response surface modeling. MCL-1/BAK complex inhibition was tested in this ALL by co-IP/immunoblot analysis. Cell death and ATG were studied in obatoclax treated RS4:11 and/or SEM-K2 cells by PI flow, LC3 and p62 Western blot analysis and EM, using doxorubicin as a control for apoptosis. Gene expression changes after vehicle treatment vs. obatoclax treatment at the IC50 and IC90 for 6 h were studied using Affymetrix HG_U133 Plus2.0 arrays. Differentially expressed genes overall and those specifically associated with ATG were queried by ANOVA (p&lt;0.01, ≥50% change in mean expression considered as significant). Q-RT PCR analysis of basal expression levels of select ATG genes (BECN1, WIPI1, MAP1LC3B) was performed in 10 of the 17 primary cases and correlations with the IC50 values were determined using Pearson correlation coefficients and their levels of significance. Changes in the expression patterns of these genes after vehicle treatment vs. treatment with obatoclax at the IC50 for 6 h and 48 h were compared in 6 cases by Q-RT PCR and cluster analysis. Results: MLL partner genes were AF4, ENL and other in 6, 5 and 1 ALL, respectively; AF9 in 2 and AF6 in 1 AML; and AF4 and ENL in 1 each bilineal leukemia. The single agent IC50’s of obatoclax suggested greater sensitivity in ALL (13–834 nM; median 104 nM) than AML (243–488nM; median 341 nM), and were 79 nM and 508 nM in the bilineal leukemias. In addition to synergy with ARAC, ADR, VP16 and DEX (Zhang AACR 2007) there was synergy with LASP and VCR in the primary MLL/AF4+ ALL. In the same ALL obatoclax decreased MCL-1/BAK dimers, suggesting interaction with the MCL-1 target, and obatoclax increased high molecular weight MCL-1/BAK complexes and decreased MCL-1, the latter of which would also decrease MCL-1/BAK dimers. Obatoclax treatment of RS4:11 and SEM-K2 cells increased PI staining and LC-3I to LC-3II conversion; EM analysis of SEM-K2 cells revealed phagophores, autophagosomes and autophagolysosomes indicative of ATG induction. Lack of p62 accumulation showed that ATG was not blocked. EM findings of apoptosis occurred in SEM-K2 cells after doxorubicin exposure. ATG was not the most affected pathway in microarray analyses of obatoclax treated SEM-K2 and RS4:11 cells, but specific analysis of ATG-related genes showed WIPI1 and MAP1LC3B upregulation. Moreover, the basal expression of BECN1 was positively correlated with obatoclax activity in the 10 primary cases (r =0.659; p=0.038), which is consistent with importance of this pathway in the drug response. ATG gene expression analysis in obatoclax treated primary MLL+ cases identified 2 patient clusters; in one cluster obatoclax decreased BECN1 and increased WIPI1 expression; in the other ATG gene expression changes were more variable. Conclusions: Obatoclax induces cell death in MLL+ leukemias via the ATG pathway even though they are apoptosis competent. This is distinct from the apoptosis activation in other cancer cell types and indicates that the targets of obatoclax are disease-specific. The activity in a broad spectrum of MLL+ leukemias indicates that obatoclax is a promising molecularly targeted agent for this population.

Blockade of IGF-1R with OSI-906 Overcomes Bortezomib-Resistance in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 925-925 ◽  
Author(s):  
Deborah J. Kuhn ◽  
Hua Wang ◽  
Richard Julian Jones ◽  
Chad C. Bjorklund ◽  
Robert Z. Orlowski
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Viable Cell ◽  
Resistant Cell ◽  
Parp Cleavage ◽  
Cell Populations ◽  
Dose Dependent

Abstract Abstract 925 Blockade of IGF-1R with OSI-906 Overcomes Bortezomib-resistance in Multiple Myeloma Deborah J. Kuhn, Hua Wang, Richard J. Jones, Chad C. Bjorklund, Robert Z. Orlowski The Department of Lymphoma & Myeloma, The University of Texas M. D. Anderson Cancer Center, Houston, TX Intro: Bortezomib (Velcade®) therapy is now one of the standards of care in the treatment of newly diagnosed and relapsed/refractory multiple myeloma. Unfortunately, like many other novel agents, the emergence of drug resistance often results in a reduced response to any subsequent therapies that contain bortezomib. Identifying the molecular signaling pathways predominant in bortezomib-resistance can lead to the discovery of therapies that can overcome or prevent the emergence of such resistance all together. Design: In order to improve our understanding of the mechanisms responsible for bortezomib-resistance, our group has developed cell line models of interleukin (IL)-6-dependent and –independent bortezomib-resistant multiple myeloma. Results: Gene expression profiling identified insulin-like growth factor (IGF-1) signaling as one pathway that was induced in bortezomib-resistant myeloma cell lines. Its role was validated in molecular studies that showed exogenous IGF-1 protected drug-naïve cells from bortezomib, while shRNA-mediated knockdown of the IGF-1 receptor (IGF-1R) in bortezomib-resistant models restored sensitivity to this proteasome inhibitor. We then evaluated whether targeting IGF-1R with the clinically relevant inhibitor OSI-906 (OSI Pharmaceuticals, Inc.) could be a valid strategy to overcome bortezomib-resistance. OSI-906 alone preferentially induced cell death in bortezomib-resistant cell lines, while drug-naïve cell populations were relatively spared. Simultaneous addition of bortezomib and increasing concentrations of OSI-906 enhanced the amount of cell death. Also, OSI-906 preferentially induced apoptosis as measured by Annexin V staining. Knockout of IGF-1R gene expression using lentiviral shRNAs in bortezomib-resistant cell lines decreased sensitivity to OSI-906 compared to their scrambled control counterparts, underscoring the importance of IGF-1R signaling in these cells. Conversely, lentiviral overexpression of IGF-1R in drug-naïve wild-type cell lines reduced apoptosis when these models were exposed to OSI-906. Next, we determined whether OSI-906 alone would have an effect on MDA-MM-002, a cell line developed from the pleural effusion of a patient with advanced myeloma. MDA-MM-002 cells, which are resistant to a number of chemotherapeutics, including bortezomib, showed no decrease in viable cell populations when treated with OSI-906 alone. However, when combined with bortezomib, there was a dose-dependent decrease in the viable cell population. Importantly, flank xenograft models of bortezomib-resistant myeloma cells in immunodeficient mice remained insensitive to bortezomib treatment, but showed a dose-dependent response to OSI-906 as evidenced by decrease tumor cell growth, and caspase-3 and PARP cleavage. Examination of synergy profiles using isobologram analysis demonstrated a high degree of synergy with OSI-906 and bortezomib over the use of either drug alone. Conclusions: In addition to the important role of IGF-1 in myeloma biology, the findings herein provide an excellent rationale for using OSI-906 to target IGF-1 signaling in combination with bortezomib as an approach to overcome, or possibly even prevent outgrowth of resistance to bortezomib in myeloma patients. Disclosures: No relevant conflicts of interest to declare.

PI3K/AKT/mTOR Signaling Is a Significant Druggable Pathway In Infant Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1669-1669 ◽  
Author(s):  
Karen A. Urtishak ◽  
Wang Li-San ◽  
David T. Teachey ◽  
Tasian K. Sarah ◽  
Jeffrey S. Barrett ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Lines ◽  
Expression Profiles ◽  
Leukemia Cell ◽  
Gene Expression Profiles ◽  
Cell Killing ◽  
Mtor Signaling ◽  
Cell Death Pathway ◽  
Mtt Assays

Abstract Introduction Infant acute lymphoblastic leukemia (ALL) is an orphan disease with unmet need for safe effective therapies. This is an urgent problem because conventional chemotherapies are ineffective and have life-threatening toxicities in infants. Although the MLL rearrangements occurring in 75% of cases are associated with poor outcome, survival is inferior whether MLL is rearranged or not. We recently reported that infant ALL proved sensitive to obatoclax mesylate (GeminX Pharmaceuticals; now an indirect, wholly owned subsidiary of Teva Pharmaceutical Industries Ltd.) in vitro regardless of poor prognostic features including MLL gene rearrangement. Moreover, we showed that the leukemia cell killing by obatoclax involved apoptosis, necroptosis and autophagy (Urtishak et al., Blood 2013). Therefore, the recent pharmaceutical abandonment of obatoclax led us to search for similarly acting drugs, the Results of which identified the well-known antipsychotic thioridazine as a candidate for potential repurposing. Methods Correlative analyses were performed between basal gene expression profiles at leukemia diagnosis and single agent obatoclax EC50 values from MTT assays in 47 cases of infant ALL from the Children's Oncology Group P9407 trial (25 MLL-AF4; 8 MLL-ENL; 7 other MLL-rearranged; 7 MLL-germline) in order to find a priori determinants of obatoclax sensitivity; significant genes were further studied by Ingenuity Pathway Analysis (IPA). A search for similarly acting compounds was conducted by Connectivity Map analysis of gene expression profiles of MLL-AF4 ALL cell lines after obatoclax treatment. MTT assays without and with cell death pathway inhibition, Western blot and flow cytometric cell death assays, and phosphoflow cytometric signaling analyses were utilized to investigate activity and target modulation by potential candidates. Results IPA identified significant correlations between basal gene expression of the mTOR and downstream intersecting eIF4/p70S6K signaling programs and obatoclax EC50 in all 47 primary cases of infant ALL, as well as in the subset of the 25 cases with MLL-AF4 rearrangements. Consistent with the relevance of this pathway in leukemia cell killing that was suggested by the basal gene expression profiles in the primary cases, the Connectivity Map analysis of obatoclax-treated cell lines for compound matching returned a number of highly ranked PI3K/AKT/mTOR signal transduction inhibitors as potential obatoclax substitutes. Three of the compounds (LY294002, wortmannin, thioridazine) were not only cytotoxic in MLL-AF4 ALL cell lines, but also they abrogated PI3K/AKT/mTOR signaling as indicated by robust inhibition of phosphorylated S6. Of these compounds, the phenothiazine derivative thioridazine, which has been used clinically for decades as a neuroleptic, was of high interest because of potential advantages of drug repurposing for more rapid drug advancement. Moreover, detailed flow cytometric and Western blot analyses, and MTT assays of thioridazine in the presence of cell death pathway inhibitors validated activation of all three cell death mechanisms in the MLL-AF4 ALL cell lines similarly to obatoclax. Conclusions Thioridazine is a well-known antipsychotic drug that also has recently recognized properties as a PI3K/AKT/mTOR signaling inhibitor and as an inhibitor of other pathways relevant to cancer. In MLL-AF4 ALL cell lines characterized by the most common chromosomal translocation in infant ALL, single-agent thioridazine is highly cytotoxic, robustly inhibits PI3K/AKT/mTOR signaling and, moreover, like obatoclax, demonstrates activity as a multi-cell-death pathway agonist. Further preclinical studies now are warranted to determine the extent to which thioridazine inhibits PI3K/AKT/mTOR signaling and causes leukemia cell killing in primary infant ALL cells in vitro and in vivo. The repurposing strategy that this drug may allow could have promise to streamline drug development in infant ALL where the need for new therapies is so urgent. Disclosures: No relevant conflicts of interest to declare.

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