The Novel Thiosemicarbazone Derivative Coti-2 Induces Mitochondrial Apoptosis in Acute Myeloid Leukemia Cells Via a p53-Independent Mechanism

Abstract Introduction TP53 mutations are rare (< 5%) in de novo acute myeloid leukemia (AML) but if present, they are associated with a very poor prognosis (< 1% overall survival at 3 years). p53 mutations have been frequently detected in poor-prognosis patients with complex karyotype or therapy-related AML. COTI-2 is a third generation thiosemicarbazone derivative that was identified as a small molecule candidate against a diverse group of human cancer cell lines using a proprietary in silico drug screening method. COTI-2 causes cancer cell death via apoptosis. COTI-2 has been proposed to restore non-functional mutant p53 conformation to functional wild-type conformation in solid cancer cells, and it has recently entered a clinical trial in patients with advanced or recurrent gynecologic malignancies (NCT02433626). The molecular mechanisms of COTI-2 to induce apoptosis, however, remain largely unknown. In this study, we investigated activity and possible modes of action of COTI-2 against AML cells, especially focusing on its p53-independent properties. METHODS AND RESULTS A total of 10 AML cell lines were exposed to COTI-2 for 72 hours and its anti-leukemia effects were determined by viable cell counts and annexin V staining. OCI-AML2, OCI-AML3, MOLM-13, MOLM-14 and MV4;11 express wild-type (WT) p53; THP-1and Kasumi-1 express mutant (MUT) p53; KG-1, U937 and HL-60 does not express p53 protein (p53 NULL). COTI-2 inhibited leukemia cell growth and increased the percentage of annexin V-positive cells, irrespective of cellular p53 status. The IC50 values (concentration at which cell growth is inhibited by 50%) for were not statistically different between p53 WT and MUT/NULL cells (10.3 ± 4.5 nM vs 20.2 ± 11.5 nM, P = 0.44). So did the ED50 values (effective concentration inducing 50% cell killing as measured by Annexin V positivity) (115.0 ± 50.9 nM vs 237.8 ± 109.9 nM, P = 0.34). We next examined the apoptotic effect on primary AML cells from patients with AML. A total of 14 samples [11 p53 WT cases and 3 p53 MUT (C135W, R248Q and C242Y) cases] were examined. COTI-2 induced apoptosis both in p53 WT (40.3 ± 6.4% compound-specific annexin V induction) and MUT samples (48.0 ± 17.7%) (P = 0.62). To elucidate the p53-independent activity of COTI-2, mRNA expression levels of TP53 (p53), CDKN1A (p21) and BBC3 (PUMA) were determined in four AML cell lines (OCI-AML3, MV4;11, HL-60 and Kasumi-1) after exposure to COTI-2. COTI-2 did not induce TP53 or its transcriptional targets CDKN1A or BBC3 in any of the cell lines. To investigate if COTI-2 activates an intrinsic pathway to induce apoptosis in AML, we used p53 NULL HL-60 cells overexpressing BCL-2 (HL-60/BCL-2), BCL-XL (HL-60/BCL-XL) and their controls (HL-60/neo). HL-60/BCL-2 and HL-60/BCL-XL respectively expressed BCL-2 and BCL-XL at greater than 4 times higher levels than HL-60/neo. Interestingly, overexpression of either BCL-2 or BCL-XL almost completely abrogated COTI-2-induced apoptosis. Similar results were obtained in OCI-AML3/BCL-2. MCL-1 overexpression only modestly inhibited COTI-2-induced apoptosis. Involvement of the extrinsic pathway was modest as JurkatI9.2 (a Jurkat clone deficient in caspase-8) and its control showed similar sensitivity to COTI-2. COTI-2 appeared to reduce MCL-1 expression levels through mTORC1 inhibition. Finally, COTI-2 strongly synergized with the BCL-2 inhibitor ABT-199 to induce apoptosis in AML cells. Conclusion COTI-2 induces mitochondrial apoptosis in AML cells, irrespective of their p53 mutational status. COTI-2-induced apoptosis depends on BCL-2 and BCL-XL expression but not on MCL1 expression or p53 activation. COTI-2 has clinical potential in AML that often expresses high levels of MCL-1, especially in combination with BCL-2 inhibition. Disclosures Danter: Critical Outcomes: Employment.

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Clinical Impact and Therapeutic Opportunity of Insulin Receptor Substrates 1/2 in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-119156 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1512-1512

Author(s):

Juan Luiz Coelho-Silva ◽

Diego Antonio Pereira-Martins ◽

Josiane Lilian Schiavinato ◽

Eduardo Magalhães Rego ◽

João Agostinho Machado-Neto ◽

...

Keyword(s):

Cell Cycle ◽

Acute Myeloid Leukemia ◽

Cell Viability ◽

Cell Lines ◽

Myeloid Leukemia ◽

Annexin V ◽

Ros Production ◽

Insulin Receptor Substrates ◽

Induced Apoptosis ◽

Acute Myeloid

Abstract Background: The identification of biological and clinical prognostic factors in acute myeloid leukemia (AML) allowed the definition of patient subgroups and the realization of risk-adapted and targeted treatment strategies. Insulin-like growth factor 1 receptor/Insulin Receptor Substrates (IGF1R/IRS) pathway plays an important role in the development of neoplasia. IRS1/2 activates AKT/mTOR and MAPK pathways, through their interaction with PI3K and GRB2, culminating in increasing cell proliferation. NT157 is an allosteric inhibitor of IGF1R-IRS1/2 signaling that showed antineoplastic effects in preclinical studies of solid tumors. However, IRS1/2 clinical function and NT157 effects were not assessed in AML. Aims: To investigate IRS1 and IRS2 mRNA expression in AML patients and their impact in clinical outcomes, and to analyze the effects of the NT157 in AML cell lines. Material and methods: Comparison of IRS1 (probe nº 204686) and IRS2 (probe nº209184_s) expression from 581 AML patients and 8 CD34+ cells from healthy subjects were analyzed using data from Amazonia! Platform. For survival analysis, IRS1 and IRS2 mRNA expression levels from 173 AML patients (92 male - median age 58 years [range: 18-65]) were obtained from TCGA AML study available online on CBioPortal for Cancer Genomics. NB4, NB4-R2, Kasumi-1 and THP1 cell lines were submitted to NT157 (0.5, 1.0, 2.0, 4.0, 8.0 or 16 µM) 72 hours and evaluated for cell viability (MTT assay), apoptosis (Annexin V/PI), cell cycle (PI), ROS production (DCFDA), mitochondria staining (MitoTracker), and protein expression/activation (western blot). Bone marrow mononuclear cells (BMMC) were obtained from 4 AML patients at diagnosis and submitted to cytotoxic assays. Statistical analyzes were performed using ANOVA, Mann-Whitney or Kruskal-Wallis and Spearman correlation tests, as appropriate. For survival analysis, Kaplan-Meyer curves were compared with the log-rank test. Cox regression analysis was also applied. Results: IRS1 expression, but not IRS2, predicted outcomes. Reduced IRS1 expression showed poorer disease-free survival (DFS) (survival median time [MT]: 10.1 months [mos] vs. 28.4 mos, P<0.001; Hazard ratio [HR]: 0.51 [CI95:0.32 - 0.79]) and overall survival (OS) (MT: 14.5 mos vs. 27.4 mos, P=0.009; HR: 0.61 [CI95:0.42 - 0.88]). IRS1 expression independently predicted poorer DFS (HR: 0.59 [CI95: 0.36 - 0.79]; P= 0.03) using cytogenetic risk stratification, age and leukocytes as confounders. Of note, IRS1 level was positively correlated with proapoptotic CD27 (r=0.51; P<0.001) and with IL17RA (r=0.62; P<0.001) related to CD34 cell differentiation. IRS2 expression was upregulated in AML harboring t(15;17) (n=36; P<0.01) and inv(16) (n=37; P=0.01) in comparison to CD34+. In NB4, NB4-R2 and Kasumi-1 cells, NT157≥0.5µM reduced cell viability (P<0.05) and increased apoptosis (P<0.05). The mean percentage of annexin V+ cells for control, NT157 2.5, 5 and 10µM were 11, 47, 73 and 75% for NB4, 11, 41, 69 and 75% for NB4-R2 and 17, 45, 61 and 64% for Kasumi-1, respectively. In TP53-null cell line THP1, NT157 reduced cell viability at doses higher than 2µM (P<0.05) and induced apoptosis at 10µM (9.1 vs. 25%; P<0.05). NT157 induced ROS production in NB4 (fold-increase of mean fluorescence intensity [MFI]: 25.8 and 24.8), NB4-R2 (MFI: 26.7 and 31.4), Kasumi-1 (MFI: 5.8 and 6.6) and THP1 (MFI: 1.8 and 4.1) at 5 and 10µM (all P<0.05) and increased mitochondrial mass in NB4 (MFI: 3.9 and 3.7), NB4-R2 (MFI: 2.6 and 2.9), Kasumi-1 (MFI: 3.2 and 4.7) and THP-1 (MFI: 2.6 and 2.2) (all P<0.05). NT157 also modulated cell cycle progression, as evidenced by G2/M arrest in THP-1 and sub-G0/G1 in other cell lines (P<0.05). The IGF1R-IRS1/2 inhibitor NT157 reduced activation/expression of IGF1R (Tyr1135), IRS1/2 (Tyr612), AKT1/2/3 (Ser473), P70S6K (Thr421/Ser424), 4EBP1 (Thr70), ERK1/2 (Thr185/Tyr187) and induced DNA damage (increased γH2AX). NT157 did not induce autophagy, as demonstrated by non-degradation of p62 and lack of conversion of LC3BI into LC3BII in cell lines tested. NT157≥0.5 µM reduced cell viability and induced apoptosis in BMMC from 4 AML patients in a dose dependent manner (P<0.05). Conclusions: In AML, downregulation of IRS1 predicted dismal prognosis and the IGF1R-IRS1/2 inhibitor NT157 exerted an antineoplastic activity, downregulated PI3K/AKT and MAPK signaling. IRS1/2 arises as a promising therapeutic target for AML patients. Disclosures No relevant conflicts of interest to declare.

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Bcl-2 Protects against p53-Induced Apoptosis through Hdm2

Blood ◽

10.1182/blood.v112.11.5333.5333 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5333-5333

Author(s):

Line Wergeland ◽

Kevin B. Spurgers ◽

Eystein Oveland ◽

Torill Høiby ◽

Manel Cascallo ◽

...

Keyword(s):

Dna Damage ◽

Acute Myeloid Leukemia ◽

Protein Level ◽

Myeloid Leukemia ◽

Proteasome Inhibitors ◽

Wild Type ◽

Inhibitory Molecule ◽

Wild Type P53 ◽

Induced Apoptosis ◽

Acute Myeloid

Abstract Hdm2 is up-regulated in several malignancies including sarcomas and acute myeloid leukemia, where it counteracts the anti-proliferative and pro-apoptotic effect of wild type p53. The anti-apoptotic protein Bcl-2 is often elevated in many tumors with wild type p53 and serves to block p53-induced apoptosis. We demonstrate that the protein level of Hdm2 positively correlates with the level of Bcl-2 and follows the Bcl-2 level in different cell systems. Over-expression of Bcl-2 protects Hdm2 from DNA-damage induced degradation in a dose dependant manner. In addition, modulation of Bcl-2 by shRNA knockdown reduced the Hdm2 protein level in parallel. Consequently, treatment of AML cells with the Bcl-2 small inhibitory molecule HA14-1 attenuated the level of Hdm2. The Bcl-2 level, but not the DNA damage induced Hdm2 degradation, was affected by disruption of the E3 ubiquitin ligase activity of Hdm2. In addition, the DNA-damage induced Hdm2 down-regulation was blocked by disrupted E1 ubiquitin-activation, defect polyubiquitination and by proteasome inhibitors. Finally, we show that Bcl-2 protection from p53-induced cell death requires co-expression of Hdm2 in double null p53/mdm2 mouse embryonic fibroblasts. Our results indicate that Bcl-2 regulates the Hdm2 level and that Hdm2 is a key mediator in Bcl-2 inhibition of p53-induced apoptosis. This is of particular therapeutic interest for cancers displaying elevated Hdm2 and Bcl-2, like sarcoma and acute myeloid leukemia.

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Stromal CYR61 Confers Resistance to Mitoxantrone Via Spleen Tyrosine Kinase Activation in Human Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.2228.2228 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2228-2228

Author(s):

Xin Long ◽

Laszlo Perlaky ◽

Tsz-Kwong Chris Man ◽

Michele S. Redell

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Stromal Cell ◽

Chemotherapy Resistance ◽

Induced Apoptosis ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a life-threatening bone marrow malignancy with a relapse rate near 50% in children, despite aggressive chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. The factors that contribute to stroma-induced chemotherapy resistance are largely undetermined in AML. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in human AML cells. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, to study stroma-induced chemotherapy resistance. Both stromal cell lines are equally effective in protecting AML cell lines and primary samples from apoptosis induced by chemotherapy agents, including mitoxantrone, etoposide, and cytarabine. By gene expression profiling using the Affymetrix U133Plus 2 platform, we previously found that CYR61 was among the genes that were commonly upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein that is expressed at relatively low levels by AML cells, and at higher levels by stromal cells. CYR61 binds and activates integrins and enhances growth factor signaling in AML cells, and it has been associated with chemoresistance in other malignancies. Our current data provide functional evidence for a role for this protein in stroma-mediated chemoresistance in AML. First, we added anti-CYR61 neutralizing immunoglobulin (Ig), or control IgG, to AML-stromal co-cultures, treated with chemotherapy for 24 hours, and measured apoptosis with Annexin V staining and flow cytometry. In THP-1+HS-27A co-cultures treated with 50 nM mitoxantrone, the apoptosis rate was 33.0 ± 3.7% with anti-CYR61 Ig v. 16.3 ± 4.2% with control IgG; p=0.0015). Next, we knocked down CYR61 in the HS-5 and HS-27A stromal cell lines by lentiviral transduction of two individual shRNA constructs, and confirmed knockdown (KD) at the gene and protein levels for both cell lines. These CYR61-KD stromal cells provided significantly less protection for co-cultured AML cells treated with mitoxantrone, compared to stromal cells transduced with the non-silencing control. For example, the apoptosis rate for THP-1 cells co-cultured with CYR61-KD HS-27A cells was 10.8 ± 0.8%, compared to 6.8 ± 1.1% for THP-1 cells co-cultured with control HS-27A cells (p=0.02). Similar results were obtained with NB-4 AML cells. These results demonstrate that CYR61 contributes to stroma-mediated chemoresistance. CYR61 binds to integrin αvβ3 (Kireeva, et al, J. Biol. Chem., 1998, 273:3090), and this integrin activates spleen tyrosine kinase (Syk) (Miller, et al, Cancer Cell, 2013, 24:45). Using intracellular flow cytometry, we found that activated Syk (pSyk) increased in THP-1 and NB-4 cell lines, and in primary AML patient samples, upon exposure to control HS-27A cells. In primary samples, the mean fluorescence intensity (MFI) for pSyk averaged 11.7 ± 1.3 in co-culture v. 6.6 ± 0.6 for cells cultured alone (p=0.004, n=10). In contrast, pSyk did not significantly increase in AML cells co-cultured with CYR61-KD HS-27A cells (MFI for primary patient samples: 8.6 ± 0.8). This result implicates Syk as a downstream signaling mediator of CYR61. To determine the role of CYR61-induced Syk signaling in chemotherapy resistance, we treated AML-stromal cell co-cultures with 3 uM R406, a potent Syk inhibitor, or DMSO, then added 300 nM mitoxantrone, and measured apoptosis after 24 hours. In AML cells co-cultured with control HS-27A cells, mitoxantrone-induced apoptosis was significantly increased by Syk inhibition (THP-1 cells: 13.7 ± 0.7% with R406 v. 10.0 ± 0.3% with DMSO, p<0.05), consistent with reduced chemoresistance. Notably, R406 did not further increase mitoxantrone-induced apoptosis in AML cells co-cultured with CYR61-KD HS-27A stromal cells (THP-1 cells: 15.7 ± 0.2% with R406 v. 16.9 ± 0.4% with DMSO). Similar results were seen with NB-4 cells, as well. These results support the notion that CYR61 signals through the integrin-Syk pathway to protect AML cells from chemotherapy. Therefore, the CYR61 - integrin - Syk pathway may be a potential therapeutic target for overcoming stroma-induced chemotherapy resistance in AML. Disclosures No relevant conflicts of interest to declare.

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A Functional Wild-Type p53 Gene Is Expressed in Human Acute Myeloid Leukemia Cell Lines

Blood ◽

10.1182/blood.v92.8.2977 ◽

1998 ◽

Vol 92 (8) ◽

pp. 2977-2979 ◽

Cited By ~ 10

Author(s):

Trenna Sutcliffe ◽

Loning Fu ◽

Jacinth Abraham ◽

Homayoun Vaziri ◽

Samuel Benchimol

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

P53 Gene ◽

Wild Type ◽

Leukemia Cell Lines ◽

Human Acute Myeloid Leukemia ◽

Acute Myeloid ◽

Myeloid Leukemia Cell

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Inhibition of Histone Deacetylases 1 and 6 Enhances Ara-C-Induced Apoptosis In Pediatric Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood.v116.21.3275.3275 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3275-3275

Author(s):

Xuelian Xu ◽

Chengzhi Xie ◽

Holly Edwards ◽

Hui Zhou ◽

Steven Buck ◽

...

Keyword(s):

Clinical Trials ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Class I ◽

Pediatric Aml ◽

Induced Apoptosis ◽

Class I Hdacs ◽

The Impact ◽

Acute Myeloid

Abstract Abstract 3275 Acute myeloid leukemia (AML) accounts for one-fourth of acute leukemias in children, but is responsible for more than half of the leukemia deaths in this patient population. Resistance to cytarabine (ara-C)-based chemotherapy is a major cause of treatment failure in this disease. Therefore, new therapies for children with AML are urgently needed. Among the newer agents that have been recently investigated in high-risk AML in adults, histone deacetylase (HDAC) inhibitors [HDACIs, e.g., valproic acid (VPA) and Vorinostat (SAHA)] are particularly notable. The ability of HDACIs to induce cell differentiation, cell cycle arrest, and apoptosis in human leukemic cells, but not in normal cells, has stimulated significant interest in their potential as anti-leukemia agents. Numerous HDACIs have been developed during the last decade and the majority of these are in clinical trials including the novel class I-selective HDACIs, MS-275 and MGCD0103, and pan-HDACIs, LBH-589 and PXD101. Despite the well-characterized molecular and cellular effects of HDACIs, single-agent activity for this class of drugs has been modest. However, the clinical usefulness of HDACIs may be increased through rationally designed combination strategies including HDACIs with standard chemotherapy drugs. We previously hypothesized that VPA synergizes with ara-C, resulting in enhanced antileukemic activity in pediatric AML, by inducing apoptosis. We examined the impact of VPA on ara-C cytotoxicities in a panel of pediatric AML cell lines and diagnostic blast samples from children with de novo AML and demonstrated highly synergistic antileukemic activities of combined ara-C and VPA. This was especially pronounced in samples with t(8;21). Our mechanistic studies revealed that induction of DNA damage and Bim underlay the synergistic antileukemic activities of this drug combination. The present study was designed to identify members of the HDAC family which were deteminants of ara-C sensitivities, and to select the optimal HDACIs that were most efficacious when combined with ara-C for treating AML. Expression profiles of HDACs 1–11 in 4 clinically relevant pediatric AML cell lines (THP-1, Kasumi-1, MV4-11, and CMS) suggested that HDACs 5 and 11 were likely not involved due to marginal or lack of expression. The remaining class II HDACs and the entire class I enzymes could be relevant to HDACI anti-leukemic activities, based on the relationships between HDAC levels and HDACI cytotoxicities and responses to the combined VPA and ara-C, although the impact of class I HDACs seemed to predominate. Treatment of THP-1 cells with structurally-diverse HDACIs [SAHA (a pan-HDACI), VPA (a relatively class I selective-HDACI), and MS-275 (a class I selective-HDACI)] and enzymatic assays following immunoprecipitation of class I HDACs, revealed that inhibition of class I HDACs could augment ara-C-induced apoptosis. However, class II HDACs (e.g., HDAC6) were also implicated since SAHA was also effective. shRNA knockdown of HDACs 1 or 6 resulted in ∼2-fold increased apoptosis induced by ara-C in THP-1 AML cells (p<0.05). This was accompanied by substantially increased expression of Bim (2.3- and 1.4-fold, respectively). Down-regulation of HDAC2 resulted in ∼30% decreased ara-C-induced apoptosis. In contrast, shRNA knockdown of HDACs 3 and 4 had no effects on ara-C-induced apoptosis in THP-1 cells. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced ara-C-induced apoptosis in pediatric AML sublines including THP-1 and Kasumi-1. Our results further establish that HDACs are promising therapeutic targets for treating pediatric AML and identified HDACs 1 and 6 as the most relevant drug targets. Accordingly, treating pediatric AML patients with pan-HDACIs may be more beneficial than HDAC isoform-specific drugs. Based on our results, incorporation of pan-HDACIs (e.g., LBH-589 and PXD101) into ara-C-based clinical trials for treating pediatric AML should be strongly considered. Disclosures: No relevant conflicts of interest to declare.

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Small Molecule Inhibition of SIRT Activity: A Novel Therapeutic Approach for Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.84.84 ◽

2011 ◽

Vol 118 (21) ◽

pp. 84-84

Author(s):

Steffan T Nawrocki ◽

Claudia M Espitia ◽

Kevin R. Kelly ◽

William G. Bornmann ◽

Jennifer S Carew

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Small Molecule ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Clonogenic Survival ◽

Altered Gene ◽

Induced Apoptosis ◽

Acute Myeloid ◽

Novel Therapeutic

Abstract Abstract 84 New therapeutic strategies are urgently needed to improve clinical outcomes for patients with acute myeloid leukemia (AML), which is an extremely aggressive disease with very few long-term survivors. The sirtuin deacetylases (SIRTs) are critical regulators of genes that are essential for longevity, cell growth, tumor suppression, and apoptosis. Elevated SIRT expression has been reported in several types of cancer and may promote pathogenesis and drug resistance by increasing the lifespan and survival capacity of malignant cells. Our preliminary analysis of SIRT expression indicated that SIRT1 was consistently expressed at significantly higher levels in AML cell lines and primary AML blasts as compared with normal controls. In order to investigate the potential role of SIRT1 as a regulator of AML pathogenesis, we utilized shRNA to stably knockdown its expression in MV4-11 and KG-1 AML cells. Cells with targeted SIRT1 knockdown displayed an altered gene expression profile as compared with non-targeted controls. Moreover, antagonizing SIRT1 expression significantly impeded the progression of AML in a xenograft mouse model. A number of deacetylase inhibitors have been clinically evaluated for cancer therapy. However, disrupting SIRT function as an anticancer strategy remains to be rigorously investigated as none of these previously studied drugs significantly inhibit the activity of this class of NAD+-dependent deacteylases. Tenovin-6 is a novel small molecule SIRT inhibitor. We investigated the efficacy and pharmacodynamic effects of tenovin-6 in AML cell lines, primary blasts from patients with AML, and mouse models. Treatment with tenovin-6 induced apoptosis and dramatically diminished AML clonogenic survival. Tenovin-6 promoted a dose-dependent increase in the acetylated levels of the SIRT-regulated gene p53 in AML cells and triggered the induction of several p53 transcriptional targets including p21 and PUMA. Targeted knockdown of PUMA with shRNA significantly reduced the pro-apoptotic effects of tenovin-6, indicating that it is a critical mediator of its anti-leukemic activity. Notably, administration of tenovin-6 to mice implanted with AML cells was well-tolerated and led to a highly significant reduction in disease burden and increase in overall survival. Our collective findings demonstrate that SIRT1 is a promising novel therapeutic target in AML. Further investigation aimed to elucidate the safety, efficacy, and mechanism of action of tenovin-6 is warranted. Disclosures: No relevant conflicts of interest to declare.

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Dual PI3K/Akt/mTOR Pathway Inhibitor Potentiate Cytarabine Cytotoxicity For Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.1289.1289 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1289-1289

Author(s):

Eun Kyung Kim ◽

Hyoung Jin Kang ◽

Hyung Joon Kim ◽

Ji Won Lee ◽

Hyery Kim ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Growth ◽

Cell Lines ◽

Myeloid Leukemia ◽

Signal Transmission ◽

Mtor Pathway ◽

Leukemic Cells ◽

Combination Regimen ◽

Dual Inhibitor ◽

Acute Myeloid

Abstract Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signal transduction pathway integrating signals from multiple receptor tyrosine kinases has been firmly established as a major determinant for cell growth, proliferation, and survival in a wide array of solid cancers. PI3K/Akt/mTOR pathway is frequently activated in acute myeloid leukemia (AML) cells and contributes to survival and drug-resistance of AML through various mechanisms. BEZ235 is one of the most promising dual inhibitor of PI3K and mTOR currently under clinical development in solid tumor area. In this study, the potential of BEZ235 was investigated as antileukemic agent using alone or with cytarabinearabinoside (AraC) as combination regimen. AML cell lines KG-1, MV 4-11, THP-1 and HL60 were treated with AraC, BEZ235 and combination regimen with various mixed ratio. BEZ235 effectively inhibited leukemic cell growth with similar range of half maximal inhibitory concentration (IC50) values among different cell lines. Apoptosis was induced gradually as BEZ235 concentration increased, but significant level of apoptosis was not shown even at higher concentration beyond IC50 value. Then, AraC-resistant MV4-11 and THP-1 cell lines were chosen to investigate interaction between BEZ235 and AraC. Using CalcuSyn software based on Chou and Talalay analysis, Combination Index (CI) value was calculated in each combination regimen. Moderate to strong synergism was shown and it was well maintained as combination ratio of AraC versus BEZ235 gradually decreased from 20:1 to 1,000:1. BEZ235 reduced resistance to AraC when it was added as combination regimen, and significance of combination effect changed according to AraC concentration. When antileukemic effect was compared among combination regimens with different schedules, synergism was maximized when BEZ235 was pretreated before AraC administration. This means BEZ235 sensitizes leukemic cells to apoptotic effect of AraC. Genetic alteration in PI3K/Akt/mTOR pathway is an attractive target to investigate and dual pathway inhibitor BEZ235 has potential to maximize AML treatment through sensitization of leukemic cells to conventional drug. The results of this experiment suggest the possibility of designing early clinical trials for the therapy - AraC administration after dual inhibitor BEZ administration. An effective control of a signal transmission system using innovate new drugs along with an accurate understanding of a signal transmission system which is the target of therapy will establish a foothold for the development of AML therapy. Disclosures: No relevant conflicts of interest to declare.

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Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways

Blood ◽

10.1182/blood.v96.12.3907.h8003907_3907_3914 ◽

2000 ◽

Vol 96 (12) ◽

pp. 3907-3914 ◽

Cited By ~ 12

Author(s):

Masao Mizuki ◽

Regina Fenski ◽

Hartmut Halfter ◽

Itaru Matsumura ◽

Rainer Schmidt ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Colony Formation ◽

Myeloid Leukemia ◽

Map Kinases ◽

Rapid Development ◽

Dominant Negative ◽

Wild Type ◽

Flt3 Mutations ◽

Induced Apoptosis ◽

Acute Myeloid

Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation of STAT5. The relative importance of the STAT5 and Ras pathways for ITD-induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony formation by 50%. Despite its weak constitutive activation by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD–mediated colony formation. Taken together, Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways.

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Induced Apoptosis Investigation in Wild-type and FLT3-ITD Acute Myeloid Leukemia Cells by Nanochannel Electroporation and Single-cell qRT-PCR

Molecular Therapy ◽

10.1038/mt.2016.6 ◽

2016 ◽

Vol 24 (5) ◽

pp. 956-964 ◽

Cited By ~ 5

Author(s):

Keliang Gao ◽

Xiaomeng Huang ◽

Chi-Ling Chiang ◽

Xinmei Wang ◽

Lingqian Chang ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Single Cell ◽

Myeloid Leukemia ◽

Leukemia Cells ◽

Wild Type ◽

Qrt Pcr ◽

Acute Myeloid Leukemia Cells ◽

Induced Apoptosis ◽

Acute Myeloid

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FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells

Blood ◽

10.1182/blood-2003-06-1969 ◽

2004 ◽

Vol 103 (1) ◽

pp. 267-274 ◽

Cited By ~ 142

Author(s):

Rui Zheng ◽

Mark Levis ◽

Obdulio Piloto ◽

Patrick Brown ◽

Brenda R. Baldwin ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Conditioned Media ◽

Autocrine Signaling ◽

Wild Type ◽

Acute Leukemias ◽

Type Receptor ◽

Acute Myeloid

Abstract The FLT3 receptor tyrosine kinase is highly expressed in most acute leukemias and frequently mutated in acute myeloid leukemia (AML). The mutated form of the receptor is constitutively activated and known to play an important role in AML, but the activation state of the overexpressed wild-type (wt) receptor is, at present, unknown. In this study, we examined the activation state of the wild-type receptor in AML. We found that the wild-type receptor was constitutively phosphorylated/activated in 8 of 12 primary AML samples and 4 of 13 leukemia cell lines. To explain why wtFLT3 is often activated, we investigated the expression of its ligand, FL, by these same cells. Coexpression of FL with FLT3 was a universal finding in both primary AML samples and leukemic-derived cell lines. To further prove that autocrine signaling was accounting for the activation, we showed that conditioned media but not fresh media was able to activate FLT3. In addition, an antibody that blocks binding of ligand to the receptor blocks FLT3 activation. Finally, depletion of FL from conditioned media is able to block the activation of FLT3. Taken together, these findings represent strong evidence that wtFLT3 is often constitutively activated in AML and thus, like its mutated form, might contribute to the altered signaling that characterizes leukemogenesis.

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