The Multi-Kinase Inhibitor TG02 targets CD34+CD38-CD123+ AML Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1823-1823
Author(s):  
Monica Pallis ◽  
Francis Burrows ◽  
Nigel H. Russell
Keyword(s):  
Kinase Inhibitor ◽  
In Vitro Toxicity ◽  
The Novel ◽  
In Vivo Models ◽  
Erk Activation ◽  
Akt Phosphorylation ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Abstract Abstract 1823 In clinical trials, FLT3 inhibitors are reported to kill circulating AML blasts, but the bone marrow is protected. We have previously reported that niche-like conditions (fibronectin and a cytokine cocktail) significantly reduced the in vitro toxicity of the FLT3 inhibitor AG1296 to AML cells. Moreover, the toxicity of AG1296 to the chemoresistant leukaemic CD34+CD38-CD123+ subset was completely abolished under niche-like conditions. The novel multi-kinase inhibitor TG02 has selectivity against cell cycle and transcriptional CDKs and JAK2 as well as FLT3. TG02 has efficacy in in vivo models and induces apoptosis in primary AML cells. We have now evaluated its in vitro toxicity under niche-like conditions in bulk AML cells and in the CD34+CD38-CD123+ subset. In a cohort of six FLT3-ITD and five FLT3-wildtype samples, 100nM TG02 induced decreases of 30% in bulk cells and 32% in CD34+CD38-CD123+ cells, whereas AG1296 (5μM) induced a median 21% decrease in bulk cells under niche-like conditions but a 0% decrease in CD34+CD38-CD123+ cells. Lestaurtinib, sorafenib and sunitinib were used as comparators (all at 100 nM) and induced, respectively, 13%, 4% and 13% decrease in bulk cells and 10%, 0% and 8% decrease in CD34+CD38-CD123+ cells. FLT3 wildtype as well as ITD samples were targeted. In order to establish the molecular pathways involved in niche-mediated chemoresistance and its reversal, we treated primary AML samples with TG02 or AG1296 for 3 hours in the presence and absence of niche proteins; we measured activating phosphorylations of STAT3 (tyr705), STAT5 (tyr694), ERK1/2 (thr202/tyr404) and AKT(ser473). Basal levels of activating phosphorylations were generally higher in the bulk cells than the CD34+CD38-CD123+ cells, possibly reflecting the increased quiescence of the latter subset. STAT3, STAT5 and ERK1/2 phosphorylation were reduced by TG02 to a slightly greater degree than by AG1296 in bulk cells. However, in CD34+CD38-CD123+ cells this contrast was enhanced, such that AG1296 was ineffective, whereas TG02 was at least as effective as in bulk cells. Niche-like conditions induced an increase in phosphorylation of STAT5, but not of the other proteins tested. TG02 reduced this to basal levels in both bulk cells and CD34+CD38-CD123+ cells. AG1296 partially blocked niche-induced STAT5 phosphorylation in bulk cells, but not in CD34+CD38-CD123+ cells. It had no effect on ERK signalling. AKT phosphorylation was not informative. In conclusion, TGO2 is more cytotoxic than comparatively selective FLT3 inhibitors towards CD34+CD38-CD123+ AML cells as well as bulk cells under niche conditions and the toxicity is associated with downregulation of STAT3, STAT5 and ERK activation. Disclosures: Pallis: Tragara Pharmaceuticals: Research Funding. Burrows:Tragara Pharmaceuticals: Employment.

scholarly journals A Mixed Type 1 and Type 2 Kinase Inhibitor That Overrides FLT3 F691 and D835 Resistance Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1074-1074 ◽  
Author(s):  
Jack Lin ◽  
Ying Zhang ◽  
Bernice Matusow ◽  
Adam Mumy ◽  
Garson Tsang ◽  
...  
Keyword(s):  
Mixed Type ◽  
Kinase Inhibitor ◽  
Binding Mode ◽  
Resistance Mutations ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Abstract Introduction: Mutations in FLT3, a class III receptor tyrosine kinase predominantly expressed on hematopoietic progenitor cells, represent the most common genetic alteration in patients with acute myeloid leukemia (AML). Approximately 25% of patients with AML harbor internal tandem duplications (ITD) within the juxtamembrane domain of FLT3. Quizartinib, the first selective FLT3 tyrosine kinase inhibitor (TKI) to show clinical activity, frequently achieves a composite complete remissions (CRc) rate of approximately 50% in relapsed and refractory FLT3-ITD+AML patients. However, patient samples obtained at the time of relapse with quizartinib revealed resistance-conferring FLT3 mutations at two kinase domain residues (F691L and D835V/Y/F). The crystal structure of the FLT3-quizartinib complex indicates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and with F830 within the DFG motif in the activation loop (A-loop). This reliance makes quizartinib responses critically vulnerable to gatekeeper (F691) and A-loop (D835) substitutions. This has led to the identification of TKIs that have activity against quizartinib-resistant FLT3 mutants, including type 1 inhibitors (e.g. crenolanib, ASP2215) targeting specifically D835 mutants, and type 2 inhibitors (e.g. pexidartinib, ponatinib) active against F691L. Until now, no FLT3 inhibitor has demonstrated potent inhibition of both classes of resistance mutations. Methods: To identify FLT3 inhibitors with activity against both gatekeeper and A-loop mutants, we used a modular approach to systematically alter the constituents of quizartinib to optimize interactions with the mutant proteins guided by X-ray structures and modeling. The compounds were profiled through a panel of in vitro growth assays and in vivo studies using Ba/F3 cells engineered to express FLT3-ITD/F691L and FLT3-ITD/D835Y, and MOLM14 quizartinib-resistant clones (MOLM14/F691L and MOLM14/D835Y) that have acquired the same mutations after chronic exposure to quizartinib. When injected into the tail vein of nude mice, the engineered Ba/F3 cells home to the spleen and proliferate, causing marked splenomegaly, whereas MOLM14 parental cells and MOLM14 resistant clones significantly reduce the overall survival of mice. Ba/F3-induced splenomegaly is directly dependent on the activity of the expressed mutant FLT3 and inhibition of splenomegaly measures target engagement in vivo. Analysis of efficacy in the MOLM14 survival models was done by Kaplan-Meier estimates and the logrank statistic. Results: Structure-guided lead optimization generated a series of FLT3 inhibitors with a mixed type 1 and type 2 binding mode and equal potency against the two classes of resistant mutants. Further optimization to improve pharmaceutical properties and maximize in vivo efficacy led to development candidate PLX3623. PLX3623 potently inhibited the growth of two Ba/F3 cell lines expressing F691L and D835Y mutants, with IC50 values of 0.18 (±0.04) and 0.15 (±0.03) nM, respectively. PLX3623 had a comparable antiproliferative effect on the MOLM14 resistant clones, with IC50values of 0.52 (±0.14) and 0.26 (±0.08) nM for the F691L and D835Y clones, respectively. In vivo PLX3623 treatment resulted in dose-dependent reduction in Ba/F3-induced splenomegaly, achieving >90% inhibition at 3 mg/kg dose. PLX3623 at 3mg/kg increased the length of survival of MOLM14 parental cell recipients by 120%, compared to the 80% increase afforded by 3 mg/kg quizartinib. At 3mg/kg dose, PLX3623 extended the survival of mice that received the two MOLM14 resistant clones by 80%; quizartinib at the same dose provided no survival benefit. Conclusion: Clonal evolution and tumor heterogeneity present a major challenge to targeted therapy and personalized cancer medicine. While acquired mutations individually may be actionable targets, often multiple inhibitors are required to combat the clonal diversity in a refractory setting. PLX3623, a FLT3 inhibitor with a mixed type 1 and type 2 binding mode, is the first TKI that is active against mutations conferring resistance through independent structural mechanisms. The in vitro and in vivo potency and favorable nonclinical safety profile of PLX3623 support its clinical development as a potentially best-in-class therapy to overcome clinical resistance to first generation FLT3 inhibitors. Disclosures Lin: Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. Matusow:Plexxikon Inc.: Employment. Mumy:Plexxikon Inc.: Employment. Tsang:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. Powers:Plexxikon Inc.: Employment. Spevak:Plexxikon Inc.: Employment. Severson:Plexxikon Inc.: Employment. Tsai:Plexxikon Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment.

scholarly journals Antileukemic effects of the novel, mutant FLT3 inhibitor NVP-AST487: effects on PKC412-sensitive and -resistant FLT3-expressing cells

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 5161-5170 ◽  
Author(s):  
Ellen Weisberg ◽  
Johannes Roesel ◽  
Guido Bold ◽  
Pascal Furet ◽  
Jingrui Jiang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Chemotherapeutic Agents ◽  
In Vivo Model ◽  
Protein Kinase Activity ◽  
The Novel ◽  
Inhibition Of Proliferation ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Abstract An attractive target for therapeutic intervention is constitutively activated, mutant FLT3, which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing, and which is currently in late-stage clinical trials. However, the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel, structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here, we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487, which selectively targets mutant FLT3 protein kinase activity, is also shown to override PKC412 resistance in vitro, and has significant antileukemic activity in an in vivo model of FLT3-ITD+ leukemia. Finally, the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus, we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target, and which could potentially be used to override drug resistance in AML.

scholarly journals ATM/G6PD-driven redox metabolism promotes FLT3 inhibitor resistance in acute myeloid leukemia

2016 ◽  
Vol 113 (43) ◽  
pp. E6669-E6678 ◽  
Author(s):  
Mark A. Gregory ◽  
Angelo D’Alessandro ◽  
Francesca Alvarez-Calderon ◽  
Jihye Kim ◽  
Travis Nemkov ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Mitochondrial Oxidative Stress ◽  
Flt3 Inhibitor ◽  
A Genome ◽  
Flt3 Inhibitors ◽  
Acute Myeloid

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.

Protection Against Vascular Leakage in Vivo by a Peptide Mimetic of the Novel Tethered Ligand Generated by Non-Canonical Cleavage of Protease Activated Receptor 1 by Activated Protein C

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 497-497
Author(s):  
Laurent Burnier ◽  
Ranjeet Kumar Sinha ◽  
Eveline A. Bouwens ◽  
John H. Griffin ◽  
Laurent O. Mosnier
Keyword(s):  
Evans Blue ◽  
Activated Protein C ◽  
Vascular Leakage ◽  
Endothelial Barrier ◽  
The Novel ◽  
Akt Phosphorylation ◽  
Tethered Ligand ◽  
Protease Activated Receptor 1

Abstract Abstract 497 Activated protein C (APC) exerts cytoprotective activities on vascular endothelium that require protease-activated receptor 1 (PAR1) whereas thrombin acting via PAR1 causes endothelial disruptive, proinflammatory actions. Last year our laboratory elucidated a unique biochemical mechanism leading to the APC's cytoprotective signaling initiation, revealing that APC can cleave PAR1 at Arg46 and that a synthetic peptide, TR47, comprising PAR1 residues 47–66, stimulates signaling in endothelial cells reflected in Akt phosphorylation and anti-apoptotic activity (see Blood 2011;118:534). Here we report novel in vitro and in vivo insights concerning the downstream effects of APC-specific cleavage at Arg46. First, using the EA.hy926 endothelial cell line, we showed that TR47 induced sustained phosphorylation of glycogen synthase kinase 3 beta (GSK3beta) at Ser9 starting at 30 min. Moreover, the TR47 time-course was similar to Akt phosphorylation. A scrambled control peptide (scrTR47) was unable to induce GSK3beta phosphorylation. TR47-induced phosphorylation of GSK3beta was inhibited by the PAR1 antagonist SCH79797, indicating that TR47-induced signaling required PAR1. Cleavage of PAR1 at Arg41 by thrombin induces phosphorylation of extracellular-regulated kinase (ERK1/2). TRAP peptide (TFLLRNPNDK), the canonical PAR1 agonist, induced strong and immediate phosphorylation of ERK whereas neither TR47 nor scrTR47 induced ERK phosphorylation. In contrast, treatment of EA.hy926 endothelial cells with TRAP did not result in phosphorylation of Akt at Ser473 or GSK3beta at Ser9. In agreement with peptides data, thrombin did not induce Akt or GSK3beta phosphorylation whereas APC did so. Thus, PAR1 cleavage at Arg46 results in phosphorylation of Ser473-Akt and Ser9-GSK3beta, whereas cleavage of PAR1 at Arg41 results in phosphorylation of ERK1/2. Activation of PAR1 by thrombin results in Ras homolog gene family member A (RhoA) activation and disruption of the endothelial barrier. In contrast, activation of PAR1 with APC results in activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and endothelial barrier protection. Using active Rac1 pulldown assays with p21-activated kinase (PAK-1)-conjugated beads and quantifying the ratio of active Rac1 over total Rac1, we showed that both TR47 and APC, but not scrTR47, activated Rac1. In an endothelium barrier transwell assay using Evans Blue to quantify thrombin-induced leakage, TR47 and APC, but not scrTR47 or TRAP, decreased vascular permeability by 40% (P < 0.05). Thus, cleavage of PAR1 at Arg46 but not at Arg41 results in endothelial barrier protective effects in vitro. To test whether TR47 also reduces vascular leakage in vivo, we setup a novel modification of the modified Miles assay to assess the effect of TR47 on VEGF-induced vascular leakage in the skin. Immunocompetent SKH1 hairless mice were used to avoid the need for hair removal that often can result in artifactual leakage due to inflammation of the skin. Evans Blue was injected intravenously followed 30 min later by 2μg of recombinant mouse APC, 125 μg of TR47 or PBS (i.v.). Recombinant VEGF-165 (75 ng, subcutaneous) or vehicle (BSA) was injected thereafter on the abdomen. After 30 min mice were placed on the Odyssey infrared fluorescence Imager and the total amount of vascular leakage was quantified as the amount of Evans Blue accumulated in the VEGF or BSA injection sites determined by infrared fluorescent at 700 nm. APC decreased leakage by 50%. TR47, but not scrTR47, injected 30 min before VEGF decreased vascular leakage by 45% (P < 0.05, n = 6 mice) compared to PBS control. Neither TR47 nor scrTR47 affected vascular leakage in the absence of VEGF. In summary, the TR47 peptide representing the sequence of the novel N-terminus that is generated by cleavage of PAR1 at Arg46 exerts remarkable biologic activities in vitro and in vivo that reflect the general cytoprotective activity profile of APC but not that of thrombin. Based on these results we propose a novel paradigm for the biochemical mechanisms of APC via PAR1 involving generation of a new N-terminal tethered ligand, which is a biased agonist that initiates APC-like cytoprotective signaling pathways. Disclosures: No relevant conflicts of interest to declare.

5009 ORAL Significant antitumour activity of the novel epothilone ZK-EPO against in vitro and in vivo models of ovarian cancer

2007 ◽  
Vol 5 (4) ◽  
pp. 313-314 ◽  
Author(s):  
S. Hammer ◽  
N. Arnold ◽  
F. Hilpert ◽  
K. Bräutigam ◽  
A. Sommer ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Antitumour Activity ◽  
The Novel ◽  
In Vivo Models

scholarly journals Minimal modification in the factor VIII B-domain sequence ameliorates the murine hemophilia A phenotype

Blood ◽  
2013 ◽  
Vol 121 (21) ◽  
pp. 4396-4403 ◽  
Author(s):  
Joshua I. Siner ◽  
Nicholas P. Iacobelli ◽  
Denise E. Sabatino ◽  
Lacramiora Ivanciu ◽  
Shangzhen Zhou ◽  
...  
Keyword(s):  
Hemophilia A ◽  
The Novel ◽  
In Vivo Models ◽  
Domain Sequence ◽  
Key Points ◽  
Minimal Modification ◽  
Therapy Strategies ◽  
Enhanced Stability

Key Points The novel FVIII variant (FVIII-RH) has enhanced stability and procoagulant activity in both in vitro and in vivo models. FVIII-RH is efficacious and safe; thus, it is an attractive molecule for protein replacement and as a transgene in gene-therapy strategies.

scholarly journals FLT3 ligand impedes the efficacy of FLT3 inhibitors in vitro and in vivo

Blood ◽  
2011 ◽  
Vol 117 (12) ◽  
pp. 3286-3293 ◽  
Author(s):  
Takashi Sato ◽  
Xiaochuan Yang ◽  
Steven Knapper ◽  
Paul White ◽  
B. Douglas Smith ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Therapeutic Strategy ◽  
Newly Diagnosed ◽  
Flt3 Ligand ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
Acute Myeloid

AbstractWe examined in vivo FLT3 inhibition in acute myeloid leukemia patients treated with chemotherapy followed by the FLT3 inhibitor lestaurtinib, comparing newly diagnosed acute myeloid leukemia patients with relapsed patients. Because we noted that in vivo FLT3 inhibition by lestaurtinib was less effective in the relapsed patients compared with the newly diagnosed patients, we investigated whether plasma FLT3 ligand (FL) levels could influence the efficacy of FLT3 inhibition in these patients. After intensive chemotherapy, FL levels rose to a mean of 488 pg/mL on day 15 of induction therapy for newly diagnosed patients, whereas they rose to a mean of 1148 pg/mL in the relapsed patients. FL levels rose even higher with successive courses of chemotherapy, to a mean of 3251 pg/mL after the fourth course. In vitro, exogenous FL at concentrations similar to those observed in patients mitigated FLT3 inhibition and cytotoxicity for each of 5 different FLT3 inhibitors (lestaurtinib, midostaurin, sorafenib, KW-2449, and AC220). The dramatic increase in FL level after chemotherapy represents a possible obstacle to inhibiting FLT3 in this clinical setting. These findings could have important implications regarding the design and outcome of trials of FLT3 inhibitors and furthermore suggest a rationale for targeting FL as a therapeutic strategy.

scholarly journals The Pim Kinase Inhibitor AZD1208 Enhances Apoptosis Induction By Clinically Active FLT3 Inhibitors in FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and in Vivo through Synergistic Downregulation of Mcl-1 and of Bcl-xL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3601-3601 ◽  
Author(s):  
Karthika Natarajan ◽  
Trevor J Mathias ◽  
Kshama A Doshi ◽  
Adriana E Tron ◽  
Manfred Kraus ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Protein Levels ◽  
Flt3 Inhibitors ◽  
Synergistic Induction ◽  
Induction Of Apoptosis

Abstract Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) are present in acute myeloid leukemia (AML) cells in 30% of cases and are associated with high relapse rate and short disease-free survival. FLT3 inhibitors have clinical activity, but their activity is limited and transient. New therapeutic approaches combining FLT3 inhibitors and inhibitors of downstream or parallel signaling pathways may increase depth and duration of responses. The Pim-1 serine/threonine kinase is transcriptionally upregulated by FLT3-ITD. We previously demonstrated that Pim-1 phosphorylates and stabilizes FLT3 and thereby promotes its signaling in a positive feedback loop. Pim kinase inhibitors are in clinical trials. Here we studied the effect of combinations of the Pim kinase inhibitor AZD1208 and clinically active FLT3 inhibitors on AML with FLT3-ITD in vitro and in vivo. Ba/F3-ITD cells, with FLT3-ITD, were grown in medium with the Pim kinase inhibitor AZD1208 at 1 μM and/or the FLT3 inhibitors quizartinib (Q), sorafenib (S) or crenolanib (C) at their IC50values of 1, 2.5 and 20 nM, respectively, and viable cells were measured at serial time points. While Q, S, C or AZD1208 treatments reduced cell numbers, compared to DMSO control, combined AZD1208 and Q, S or C treatments abrogated proliferation. Because FLT3-ITD cells remain responsive to FLT3 ligand (FLT3L) despite constitutive FLT3 activation and increased FLT3L levels following chemotherapy have been hypothesized to contribute to relapse, we repeated the proliferation experiments in the presence of 0, 1, 3 and 10 ng/ml FLT3L. FLT3L produced a concentration-dependent increase in proliferation and, while Q, S, C or AZD1208 treatments individually reduced cell numbers, combined AZD1208 and Q, S or C abrogated proliferation at all FLT3L concentrations tested, suggesting that these combinations overcome growth stimulation by FLT3L. To understand the anti-proliferative effect of combined Pim-1 and FLT3 inhibitors, we first studied cell cycle effects of AZD1208 and Q, S or C in Ba/F3-ITD cells and of AZD1208 and Q in the additional FLT3-ITD cell lines 32D-ITD, MV4-11 and MOLM14. We found a progressive increase in sub-G1 phase cells at 24, 48 and 72 hours, consistent with induction of apoptosis. Synergistic induction of apoptosis was confirmed by Annexin V/propidium iodide labeling of Ba/F3-ITD and 32D-ITD cells treated for 48 hours with AZD1208 combined with Q (p<0.0001), S (p<0.0001) or C (p<0.001), and of MV4-11 (p<0.0001) and MOLM14 (p<0.05) cells treated with AZD1208 combined with Q, in relation to each drug alone. Apoptosis was additionally confirmed by loss of mitochondrial membrane potential. Synergistic induction of apoptosis was not seen in Ba/F3-WT or 32D-WT cells, with wild-type FLT3, indicating a FLT3-ITD-specific effect. Synergistic (p<0.01) induction of apoptosis was seen in three FLT3-ITD AML patient samples treated in vitro with AZD1208 combined with Q. In an in vivo model, synergistic decrease in tumor volume was seen with combined AZD1208 and Q therapy in mice with subcutaneously implanted MV4-11 cells, with FLT3-ITD, but not with KG1a cells, with wild-type FLT3. Mechanistically, combined AZD1208 and Q treatment in vitro did not increase reactive oxygen species, compared to each drug alone, but increased both cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) levels, and caspase 3 cleavage was reduced by co-incubation with the pan-caspase inhibitor Z-VAD. Moreover, combined AZD1208 and Q treatment caused a synergistic decrease in expression of the anti-apoptotic Mcl-1 and of Bcl-xL proteins, but did not significantly alter Bim-1, p-Bad, Bad, Bax, Bak or Bcl-2, pro- and anti-apoptotic protein levels. Bcl-xL mRNA expression decreased along with protein levels, but Mcl-1 mRNA levels remain unchanged, indicating post-transcriptional down-regulation of Mcl-1 by the combination treatment. In summary, synergistic cytotoxicity of AZD1208 and clinically active FLT3 inhibitors was demonstrated in FLT3-ITD cell lines and patient samples in vitro and in cell lines in vivo, via caspase-mediated apoptosis, associated with a synergistic decrease in Mcl-1 and Bcl-xL expression. Our data suggest clinical promise for combination therapy with Pim kinase and FLT3 inhibitors in patients with AML with FLT3-ITD. Disclosures No relevant conflicts of interest to declare.

The potential toxicological insights about the anti-HIV drug azidothymidine-derived monoselenides in human leukocytes: Toxicological insights of new selenium-azidothymidine analogs

2016 ◽  
Vol 36 (9) ◽  
pp. 910-918 ◽  
Author(s):  
DOC Mariano ◽  
D de Souza ◽  
DF Meinerz ◽  
J Allebrandt ◽  
AF de Bem ◽  
...  
Keyword(s):  
Antioxidant Properties ◽  
Damage Index ◽  
Human Leukocyte ◽  
In Vitro Toxicity ◽  
Organoselenium Compounds ◽  
In Vivo Models ◽  
Human Leukocytes ◽  
Anti Hiv

Acquired immunodeficiency syndrome (AIDS) is a worldwide disease characterized by impairments of immune function. AIDS can be associated with oxidative stress (OS) that can be linked to selenium (Se) deficiency. Se is fundamental for the synthesis of selenoproteins, such as glutathione peroxidase and thioredoxin reductase. These enzymes catalyze the decomposition of reactive oxygen species and contribute to maintain equilibrium in cell redox status. Literature data indicate that organoselenium compounds, such as ebselen and diphenyl diselenide, have antioxidant properties in vitro and in vivo models associated with OS. Nevertheless, selenocompounds can also react and oxidize thiols groups, inducing toxicity in mammals. Here, we tested the potential cytotoxic and genotoxic properties of six analogs of the prototypal anti-HIV drug azidothymidine (AZT) containing Se (5′-Se-(phenyl)zidovudine; 5′-Se-(1,3,5-trimethylphenyl)zidovudine; 5′-Se-(1-naphtyl)zidovudine; 5′-Se-(4-chlorophenyl)zidovudine) (C4); 5′-Se-(4-methylphenyl)zidovudine (C5); and 5′-(4-methylbenzoselenoate)zidovudine). C5 increased the rate of dithiothreitol oxidation (thiol oxidase activity) and C2-C4 and C6 (at 100 µM) increased DNA damage index (DI) in human leukocytes. Moreover, C5 (200 µM) decreased human leukocyte viability to about 50%. Taken together, these results indicated the low in vitro toxicity in human leukocytes of some Se-containing analogs of AZT.

A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 3885-3891 ◽  
Author(s):  
Mark Levis ◽  
Jeffrey Allebach ◽  
Kam-Fai Tse ◽  
Rui Zheng ◽  
Brenda R. Baldwin ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Internal Tandem Duplication ◽  
Activating Mutations ◽  
Flt3 Inhibitor

Constitutively activating internal tandem duplication (ITD) and point mutations of the receptor tyrosine kinase FLT3 are present in up to 41% of patients with acute myeloid leukemia (AML). These FLT3/ITD mutations are likely to be important because their presence is associated with a poor prognosis. Both types of mutations appear to activate the tyrosine kinase activity of FLT3. We describe here the identification and characterization of the indolocarbazole derivative CEP-701 as a FLT3 inhibitor. This drug potently and selectively inhibits autophosphorylation of wild-type and constitutively activated mutant FLT3 in vitro in FLT3/ITD-transfected cells and in human FLT3-expressing myeloid leukemia–derived cell lines. We demonstrate that CEP-701 induces a cytotoxic effect on cells in a dose-responsive fashion that parallels the inhibition of FLT3. STAT5 and ERK1/2, downstream targets of FLT3 in the signaling pathway, are inhibited in response to FLT3 inhibition. In primary leukemia blasts from AML patients harboring FLT3/ITD mutations, FLT3 is also inhibited, with an associated cytotoxic response. Finally, using a mouse model of FLT3/ITD leukemia, we demonstrate that the drug inhibits FLT3 phosphorylation in vivo and prolongs survival. These findings form the basis for a planned clinical trial of CEP-701 in patients with AML harboring FLT3- activating mutations.

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