New Therapeutic Opportunities for Pediatric Patients with t(6;11)-Rearranged Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 749-749
Author(s):  
Claudia Tregnago ◽  
Matteo Zampini ◽  
Valeria Bisio ◽  
Barbara Buldini ◽  
Stefano Indraccolo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Ras Pathway ◽  
Primary Blast ◽  
Nsg Mice ◽  
Pharmacologically Active ◽  
Acute Myeloid

Abstract Purpose. Among pediatric acute myeloid leukemia (AML), the t(6;11)(q27;q23) MLL-AF6 translocation accounts for 26% of MLL-rearranged AML, and is associated with a worse prognosis (event-free survival of 23.3% at 3-years) compared to other forms of MLL-rearranged AML1. Gene expression profile analysis revealed a specific transcriptional signature, and this peculiarity has been explained by the mislocalization of AF6 protein into the nucleus with a consequent hyperactivation of the RAS pathway in these patients. The uncovered involvement of the RAS pathway in this AML subgroup provides the rationale for searching new therapeutical strategies to selectively target MLL-AF6-rearranged cells. Methods. We established a cell-based drug screening assay, by testing a library of 1,280 pharmacologically active compounds (Lopac library, Sigma-Aldrich) on t(6;11)-rearranged ML2 and SHI-1 cell lines. Compounds (used at 10μM) which decreased cell viability by at least 50% (by ATP measurement) were further tested in different AML cell lines (HL60, as well as NOMO1 and THP1, both t(9;11)MLL-AF9 rearranged), to exclude those with broad anti-leukemic activity and to focus specifically over MLL-AF6 action. Finally, functional studies were performed for the compounds resulted selective for the MLL-AF6 rearrangement in cell lines and patient's primary blast cultures and the most promising drug was tested in vivo using NSG mice. Results. Of 1,280 compounds, 104 and 93 impaired cell proliferation of ML2 and SHI-1, respectively. 73 were found efficacious over HL60 and, thus, excluded. Then, the remaining 20 compounds were evaluated in other MLL-cell lines (NOMO-1 and THP-1), and finally 10/20 resulted active selectively on t(6;11)-rearranged cell lines. The selected compounds were Arvanil, CP-100356 monohydrochloride, Fluspirilene, CID2858522, Eupatorin, ANA-12, BAY 61-3606 hydrochloride hydrate, Ara-G hydrate, Tyrphostin 47, Thioridazine hydrochloride and were also confirmed to impair viability over t(6;11) primary blast cultures from patients. Among them, we were particularly interested in Fluspirilene and Thioridazine, these compounds being both antipsychotics working as dopamine receptor (DR) antagonists and FDA approved. By flow cytometry we showed the DRs (DR-1 to DR-5) expression in ML2, SHI-1, NOMO-1, THP1 and SKNO-1 whereas HL60 resulted devoid of DRs. Blasts from t(6;11)-rearranged patients (n=3) expressed DRs as well. Treatment of the cell lines with Fluspirilene and Thioridazine triggered apoptosis induction in SHI-1, and to a lesser extent in ML2, due to autophagy activation, whereas no effects were observed on HL60, NOMO-1, THP1 and SKNO-1. Clonogenic assay showed that after 24 hours of treatment self-renewal ability of SHI-1 and ML-2 significantly decreased, with no effects observed in other cell lines. Same results were obtained in primary cultures from patients t(6;11), without toxic effects on healthy bone marrow cells, confirming the drug specific activity over leukemia proliferation, and with Thioridazine being more active. NSG mice were then flank injected with t(6;11) cells and treated with Thioridazine 12 mg/kg; treatment significantly inhibited tumor growth in vivo (compared to mice treated with DMSO, p<0.05). By western blot and phospho-flow cytometry analysis we explored which pathway was DRs mediated, and identified a dramatic decrease of MEK and ERK phosphorylation since 6 hours post-treatment, indicating that the RAS pathway was involved. Conclusions. This study led to the identification of DRs expression in myeloid blasts, and revealed their role in leukemia maintenance exclusively of the t(6;11)-rearranged AML. We identified a series of new compounds to be prioritized for further analysis in MLL-AML; in particular Thioridazine deserves further investigation as a novel therapeutic strategy to improve outcome of t(6;11)-rearranged patient's. 1 Pigazzi M, et al Leukemia. 2011 Mar;25(3):560-3. Disclosures Indraccolo: OncoMed Pharmaceuticals, Inc.: Research Funding.

scholarly journals Mitophagy Plays a Key Role in the Anti-Leukemic Activity of Autophagy Inhibitors Under Hypoxia in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1278-1278
Author(s):  
Hannah R.S. Fay ◽  
Kaitlyn M. Dykstra ◽  
Matthew Johnson ◽  
Tara L. Cronin ◽  
Linda Lutgen-Dunckley ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Tumor Burden ◽  
Clinical Development ◽  
Hypoxic Conditions ◽  
Nsg Mice ◽  
Acute Myeloid ◽  
Advisory Role

Background: Leukemia stem cells (LSCs) and acute myeloid leukemia (AML) blasts persisting in the bone marrow (BM) after chemotherapy are key drivers of AML relapse and chemotherapy refractoriness. The hypoxic microenvironment of the BM is known to protect these cells, however the mechanisms behind this chemoresistance are not well understood. We have previously shown that AML cell lines cultured under hypoxia upregulate autophagy. Blocking autophagy with lysosome-based autophagy inhibitor Bafilomycin A1 (Baf A1) was shown to target LSCs in colony formation (CF) assays and in vivo serial transplants in NSG mice. Another autophagy inhibitor chloroquine (CQ) also demonstrated anti-LSC effects in CF assays. The clinical development of Baf A1 and CQ has been limited due to poor pharmacokinetics and toxicity. Lys05 is a promising CQ derivative with increased potency and therapeutic potential. Recent studies have shown that LSCs have an increased reliance on oxidative phosphorylation (OXPHOS) as well as mitophagy, the autophagic degradation of damaged mitochondria. Hypoxia has also been demonstrated to increase mitophagy in certain cell types through upregulation of BNIP3. We hypothesized that AML cells cultured under hypoxia would have an increased reliance on mitophagy for mitochondrial homeostasis and survival. Treatment with autophagy inhibitors would be expected to block mitophagy and induce AML cell death specifically under hypoxia. Methods: Human AML cells (MOLM13) were cultured under hypoxia (1% O2) or normoxia (21% O2). NSG mice were inoculated with luciferase expressing MOLM13-BLIV & treated with vehicle (DMSO) or Lys05 (40 mg/kg) IP for 18 days. Tumor burden was assessed by bioluminescence. CF assays were established with AML patient cells in the presence of vehicle or Lys05 in MethoCult under normoxia/hypoxia and counted on day 13. Annexin V-FITC/PI flow cytometry was used to measure apoptosis. OXPHOS was assessed with a Seahorse XFe96 using the Mitochondrial Stress Test. Mitochondrial mass was measured by flow cytometry using MITO-ID Green. BNIP3 & PINK1 protein expression was visualized via western blot. Results: Treatment with Lys05 decreased in vivo tumor burden significantly in NSG mice systemically engrafted with human AML cells. We also saw a marked decrease in the number of CF-units in primary AML patient samples treated with Lys05 which was further enhanced under hypoxia. Treatment of AML cell lines with CQ and Lys05 also enhanced apoptosis under hypoxia as compared to normoxia. Baf A1, however, showed equal amounts of apoptosis in normoxia and hypoxia (Fig. 1A). While Baf A1, CQ, and Lys05 all inhibit autophagy through deacidification of the lysosome, Baf A1 has been shown to induce additional effects on mitochondria, inducing uncoupling of OXPHOS and depolarization. We therefore examined the autophagy inhibitors' effect on mitochondrial function. At 24 hours, Baf A1 caused a significant decrease in basal and maximal respiration under both hypoxia and normoxia. CQ and Lys05, however, only showed a significant decrease in OXPHOS under hypoxia, with no effect on mitochondrial function under normoxia. We postulated that this effect arose from increased mitophagy under hypoxia. BNIP3 expression levels were enhanced under hypoxia in MOLM13 cells at 24- 48 hours. If mitophagy is constitutively occurring, blocking this process would cause an increase in the number of mitochondria. As expected, the number of mitochondria increased when treated with Lys05 or CQ under hypoxia, but not under normoxia, suggesting that mitophagy is occurring only under hypoxic conditions. Baf A1 caused an increase in mitochondria under both hypoxia and normoxia, suggesting that Baf A1 can both induce mitophagy and block mitochondrial degradation. We further confirmed this was due to mitophagy by assessing expression of the mitophagy protein PINK1. In normoxia, Baf A1 showed an almost 2-fold increase in PINK1 compared to the vehicle whereas CQ did not (Fig. 1B). Conclusion: We have identified a class of autophagy inhibitors that displays enhanced efficacy in AML cells under hypoxic conditions that reflect the BM microenvironment. This is due in part to their ability to target AML cellular reliance on mitophagy. These results provide the rationale for the further clinical development of Lys05 or other lysosome-based autophagy inhibitors as a novel means of targeting minimal residual disease in AML therapy. Disclosures Guzman: Samus Therapeutics: Patents & Royalties: intellectual rights to the PU-FITC assay; SeqRx: Consultancy; Cellectis: Research Funding. Wang:Pfizer: Other: Advisory role, Speakers Bureau; Stemline: Other: Advisory role, Speakers Bureau; Daiichi: Other: Advisory role; Amgen: Other: Advisory role; Agios: Other: Advisory role; Abbvie: Other: Advisory role; Kite: Other: Advisory role; Jazz: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role.

scholarly journals CD37 Expression in Acute Myeloid Leukemia Provides New Target for Directed Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4056-4056
Author(s):  
Karilyn Larkin ◽  
Erin Guth ◽  
Bonnie K. Harrington ◽  
Nicole Grieselhuber ◽  
Natarajan Muthusamy ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Overall Survival ◽  
Clinical Trials ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Annexin V ◽  
Knock Out ◽  
Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is a disease for which there is an urgent need for therapy that is more effective and less toxic. Antibody based therapy has been of limited success due to both the lack of consistent immunophenotype in AML as well as the overlap with normal hematopoetic stem cells (HSCs). CD37 is a tetraspanin best characterized for its role in B-cell development and immune response (Van Spriel et al., 2004, 2009, 2012), recently shown to be expressed on AML cells but not in normal HSCs (Pereira et al.,2015). Debio 1562 (formerly IMGN529) is a potent antibody drug conjugate (ADC) directed at CD37, comprised of the antibody K7153A linked to the microtubule inhibitor (DM1) and has already demonstrated tolerability and efficacy in early phase clinical trials in lymphoid malignancies. We evaluated the expression of CD37 across primary AML samples as well as the activity of Debio 1562 in both in vitro and in vivo AML model systems. Methods: MTS assays were used to assess relative viability in AML cell lines with (THP1, OCI-AML3) or without (KG-1a) CD37 expression. Cells were treated with either the ADC, the payload conjugated to an isotype antibody (Iso-DM1) or a vehicle control for 72 hours. After determining the IC50 for each cell line, flow cytometry annexin V/PI was performed to assess apoptosis and cell death. THP-1 and OCI-AML3 CD37 knock out cell lines were created using an inducible CRISPR Cas9 system and evaluated by MTS and annexin V/PI following confirmation of knockdown of CD37 expression. Primary AML samples were obtained to assess CD37 expression by flow cytometry using K7153A-PE and separately used to evaluate direct cytotoxicity in an in vitroassay. Peripheral blood mononuclear cells from newly diagnosed AML patients were treated with either therapy, cells were harvested after 72 hours and subsequently stained with CD2/CD19/CD45 as well as EdU to assess degree of proliferation and analyzed using flow cytometry with the addition of Countbright beads to measure absolute cell counts. In vivo therapy evaluations were performed in NSG mice were engrafted with the THP1 cell line, and then treated with either the ADC, Iso-DM1 or vehicle. The mice were treated twice weekly with 10mg/kg given IP, for 7 total doses. The mice were monitored for overall survival. Results: Analysis of the TCGA database shows AML patients with higher levels of CD37 transcripts had lower overall survival (p<0.0001). MTS and Annexin V/PI assays on cell lines show selective, dose dependent cytotoxicity on CD37 expressing AML cells (Debio 1562 IC50 range 0.1-1ug/ml), with minimal cytotoxicity observed at doses up to 5ug/ml of Iso-DM1, and lack of cytotoxicity in the respective CD37 knock-out cells. Primary AML samples similarly show a selective decrease in blasts when treated with the ADC for 72 hours in culture supported with human cytokines, particularly in samples that showed a higher degree of proliferation while in culture (p=0.0156). THP1 xenograft studies showed not only a prolonged survival advantage (p<0.0001), but elimination of the disease by histopathology in animals who received treatment with Debio 1562. Conclusions: CD37 is expressed across a wide range of AML subtypes, and higher transcript levels of CD37 expression is associated with decreased survival. Despite the majority of primary samples showing minimal to modest expression of the protein when compared to B lymphocytes, CD37 is an efficient target on AML cells using Debio 1562. Additional studies are ongoing to evaluate the mechanism of CD37 trafficking in myeloid cells as well as the ability to upregulate this important target. This work provides rationale for phase I clinical trials of Debio 1562 in AML patients, as well as demonstrating CD37 as a potential target for other CD37 Immunotherapies. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

BCL-2-Selective BH3 Mimetic ABT-199 Is a Potent Agent For Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1456-1456 ◽  
Author(s):  
Rongqing Pan ◽  
LaKeisha Debose ◽  
Juliana M Benito ◽  
Leonard S Golfman ◽  
Patrick A Zweidler-McKay ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Protein Expression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Cancer Drug ◽  
Apoptosis Induction ◽  
Nsg Mice ◽  
Bh3 Mimetic ◽  
Acute Myeloid

Abstract Evasion of apoptosis is a key hallmark of cancer. BCL-2 family proteins, the central regulators of apoptosis, are often aberrantly expressed in tumors. Pro-apoptotic BCL-2 members bind and sequester anti-apoptotic BCL-2 proteins via their BH3 domains. Thus, BH3 mimetics represent a promising direction in cancer drug development. ABT-263, designed as a BH3 mimetic to inhibit BCL-2, BCL-XL, and BCL-W, has demonstrated efficacy in preclinical and clinical studies. However, thrombocytopenia is common in patients treated with ABT-263 due to the inhibition of BCL-XL, which is indispensable for platelet survival. ABT-199 (GDC-0199), a second-generation BH3 mimetic, has higher affinities for BCL-2 protein (Ki < 0.01 nM), which enhances the specificity of this agent to kill cancer cells without provoking unwanted thrombocytopenia (Souers, et al, Nature Med, 2013). Since BCL-2 is often overexpressed in hematological malignancies including acute myeloid leukemia (AML), we evaluated the anti-cancer effects of ABT-199 on AML cells. As a measure of BCL-2 specificity, BCL-XL overexpression in sensitive HL-60 cells resulted in complete resistance to ABT-199, while BCL-2 overexpression in these cells conferred moderate resistance to apoptosis induction. Moreover, OCI-AML3 cells with high MCL-1 levels were highly resistant to ABT-199, while knockdown of this protein greatly sensitized cells to this BH3 mimetic. Among 12 genetically diverse AML cell lines tested, seven were sensitive to ABT-199-induced apoptosis with 48-h EC50 ranging from 1.5 nM to 145 nM. In these seven sensitive, BCL-2 dependent cell lines, ABT-199 was uniformly more potent than ABT-737 (another BCL-2 inhibitor with a spectrum similar to ABT-263, p = 0.016). Next, we tested ABT-199 in 15 primary samples from relapsed/refractory AML patients. Twelve patient samples showed high sensitivity to apoptosis induction following 48-h exposure to ABT-199 (EC50 < 10 nM). In a larger set of 23 cryopreserved AML patient samples, including AML cells with diploid cytogenetics and mutations in FLT3, NRAS, and NPM1 genes, 18 (78%) were sensitive to ABT-199 (100 nM). However, samples from patients with complex cytogenetics, t(8;21) and JAK2 mutation (n = 12) were largely insensitive to ABT-199 (17% response rate). Interestingly, in five of six primary AML samples with high blast counts, ABT-199 induced marked apoptosis in CD34+/CD38- AML stem/progenitor cells compared to bulk AML blasts (p = 0.01). Quantitative Western blot was used to determine BCL-2 protein levels in AML cell lines. Spearman analysis showed that EC50 of ABT-199 correlated negatively with BCL-2 protein expression (r = -0.605, p = 0.0143) and correlated positively with BCL-XL protein expression (r = 0.633, p = 0.0101). Similar correlations were also observed in primary AML samples, suggesting that pre-treatment cellular BCL-2 and BCL-XL levels might have utility as predictive markers of ABT-199 sensitivity. We next examined the in vivo anti-leukemic efficacy of ABT-199 in NOD SCID gamma (NSG) mice injected with luciferase-labeled MOLM-13 cells. The mice were treated with ABT-199 by daily oral gavage (a 2-wk treatment at dose of 100 mg/kg). Bioluminescence imaging showed that ABT-199 treatment significantly inhibited leukemia burden, which was also manifested by smaller spleen size and prolonged overall survival (p = 0.0004) when compared to the vehicle-treated mice. Furthermore, a 2-wk ABT-199 treatment significantly reduced leukemia burden (> 50%) in bone marrows of NSG mice engrafted with primary FLT3-mutated AML cells (i.e., a mean of 70 ± 16% human CD45+ cells in bone marrow of control mice (n = 9) versus 32.7 ± 12% in ABT-treated mice (n = 11), p = 0.00002). Conclusions: the in vitro and in vivo efficacy data indicates that ABT-199 is a selective BCL-2 inhibitor, a potent apoptogenic agent, and hence a promising candidate for AML BCL-2-targeted therapy. Disclosures: Leverson: AbbVie, Inc.: Employment, Equity Ownership. Konopleva:AbbVie: Research Funding.

scholarly journals Anti-leukemia properties of cadmium nanoparticles in the in vitro and in vivo condition: A chemobiological study

2021 ◽  
Author(s):  
Yudi Miao ◽  
Behnam Mahdavi ◽  
Mohammad Zangeneh
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Aqueous Extract ◽  
Myeloid Leukemia ◽  
Antioxidant Properties ◽  
Leukemia Cell ◽  
Sem Images ◽  
Acute Myeloid

IntroductionThe present study investigated the anti-acute myeloid leukemia effects of Ziziphora clinopodides Lam leaf aqueous extract conjugated cadmium nanoparticles.Material and methodsTo synthesize CdNPs, Z. clinopodides aqueous extract was mixed with Cd(NO3)2 .4H2O. The characterization of the biosynthesized cadmium nanoparticles was carried out using many various techniques such as UV-Vis. and FT-IR spectroscopy, XRD, FE-SEM, and EDS.ResultsThe uniform spherical morphology of NPs was proved by FE-SEM images with NPs the average size of 26.78cnm. For investigating the antioxidant properties of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, the DPPH test was used. The cadmium nanoparticles inhibited half of the DPPH molecules in a concentration of 196 µg/mL. To survey the cytotoxicity and anti-acute myeloid leukemia effects of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, MTT assay was used on the human acute myeloid leukemia cell lines i.e., Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr. The IC50 of the cadmium nanoparticles was 168, 205, and 210 µg/mL against Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr cell lines, respectively. In the part of in vivo study, DMBA was used for inducing acute myeloid leukemia in mice. CdNPs similar to daunorubicin ameliorated significantly (p≤0.01) the biochemical, inflammatory, RBC, WBC, platelet, stereological, histopathological, and cellular-molecular parameters compared to the other groups.ConclusionsAs mentioned, the cadmium nanoparticles had significant anti-acute myeloid leukemia effects. After approving the above results in the clinical trial studies, these cadmium nanoparticles can be used as a chemotherapeutic drug to treat acute myeloid leukemia in humans.

scholarly journals The Salt-Inducible Kinase inhibitor YKL-05-099 suppresses MEF2C function and acute myeloid leukemia progressionin vivo

2019 ◽  
Author(s):  
Yusuke Tarumoto ◽  
Shan Lin ◽  
Jinhua Wang ◽  
Joseph P. Milazzo ◽  
Yali Xu ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Disease Progression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Clinical Investigation ◽  
Genetic Targeting ◽  
Acute Myeloid

AbstractLineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a Salt-Inducible Kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF MEF2C. In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. Here, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells underin vitroandin vivoconditions. Similar phenotypes were obtained when exposing cells to YKL-05-099, which caused cell cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele ofSIK3, we found that the anti-proliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated two different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progressionin vivoand extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-positive AML and provide a rationale for developing drug-like inhibitors of SIK3 for definitive pre-clinical investigation and for studies in human patients with leukemia.Key PointsAML cells are uniquely sensitive to genetic or chemical inhibition of Salt-Inducible Kinase 3in vitroandin vivo.A SIK inhibitor YKL-05-099 suppresses MEF2C function and AMLin vivo.

scholarly journals In Vivo Expansion of Co-Transplanted T Cells Impacts on Tumor Re-Initiating Activity of Human Acute Myeloid Leukemia in NSG Mice

PLoS ONE ◽  
2013 ◽  
Vol 8 (4) ◽  
pp. e60680 ◽  
Author(s):  
Malte von Bonin ◽  
Martin Wermke ◽  
Kadriye Nehir Cosgun ◽  
Christian Thiede ◽  
Martin Bornhauser ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Nsg Mice ◽  
Human Acute Myeloid Leukemia ◽  
Acute Myeloid

The Investigational Multi-Targeted Kinase Inhibitor TAK-901 Antagonizes Acute Myeloid Leukemia Pathogenesis: Results of Preclinical Studies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 581-581
Author(s):  
Patrick Griffin ◽  
Steffan T Nawrocki ◽  
Takashi Satou ◽  
Claudia M Espitia ◽  
Kevin R. Kelly ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Standard Of Care ◽  
Clonogenic Survival ◽  
Preclinical Studies ◽  
Acute Myeloid

Abstract Abstract 581 The long-term prognosis for the majority of patients diagnosed with acute myeloid leukemia (AML) is very poor due, in part, to pre-existing myelodysplasia, multidrug resistance, and co-existing morbidities that limit therapeutic options. Novel strategies are essential in order to improve clinical outcomes. TAK-901 is an investigational small molecule kinase inhibitor that is currently being evaluated in Phase I trials. In preclinical studies, TAK-901 has demonstrated significant effects against a number of kinases with important roles in cancer including the Aurora kinases, which are key regulators of mitosis and whose overexpression in cancer promotes genetic instability, malignant pathogenesis, and drug resistance. We hypothesized that simultaneously targeting the activity of the Auroras and other oncogenic kinases with TAK-901 would disrupt AML pathogenesis. In order to test our hypothesis, we investigated the efficacy and pharmacodynamic activity of TAK-901 human AML cell lines, primary AML specimens, and an orthotopic bioluminescent disseminated mouse model of AML. TAK-901 potently diminished the viability of a panel of 8 AML cell lines as well as primary cells obtained from patients with AML. Acute exposure to TAK-901 ablated clonogenic survival, triggered the accumulation of polyploid cells, and induced apoptosis. The cytostatic and cytotoxic effects of TAK-901 were associated with significantly increased expression of the cyclin-dependent kinase inhibitor p27, growth arrest and DNA-damage-inducible 45a (GADD45a), and the BH3-only pro-apoptotic protein PUMA. Chromatin immunoprecipitation (ChIP) assays revealed that the elevation in the expression of these genes caused by administration of TAK-901 was due to increased FOXO3a transcriptional activity. The in vivo anti-leukemic activity of TAK-901 was investigated in a disseminated xenograft mouse model of AML established by intravenous injection of luciferase-expressing MV4-11 cells. IVIS Xenogen imaging was utilized to monitor disease burden throughout the study. In this mouse model, administration of TAK-901 was very well-tolerated and significantly more effective than the standard of care drug cytarabine with respect to suppressing disease progression and prolonging overall survival. Analysis of specimens collected from mice demonstrated that TAK-901 inhibited the homing of AML cells to the bone marrow microenvironment and induced AML cell apoptosis in vivo. Our collective findings indicate that TAK-901 is a novel multi-targeted kinase inhibitor that has significant preclinical activity in AML models and warrants further investigation. Disclosures: Satou: Takeda Pharmaceuticals: Employment. Hasegawa:Takeda Pharmaceuticals: Employment. Romanelli:Millennium Pharmaceuticals: Employment. de Jong:Takeda San Diego: Employment. Carew:Millennium Pharmaceuticals: Research Funding.

Identifying Small Molecules That Overcome HoxA9-Mediated Differentiation Arrest in Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3513-3513
Author(s):  
David B. Sykes ◽  
Mark K Haynes ◽  
Nicola Tolliday ◽  
Anna Waller ◽  
Julien M Cobert ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Small Molecules ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Myeloid Differentiation ◽  
Prostacyclin Receptor ◽  
Acute Myeloid

Abstract Abstract 3513 AML in adults is a devastating disease with a 5-year survival rate of 25%. We lack new treatments for AML, and the chemotherapy standard of care remains unchanged in thirty years. One success story in the treatment of AML has been the discovery of drugs that trigger the differentiation of leukemic blasts in the small subset of patients with acute promyelocytic leukemia. However, differentiation therapy is unfortunately not available for the remaining 90% of non-APL acute myeloid leukemia patients. Understanding and targeting the mechanism of differentiation arrest in AML has been under investigation for more than four decades. There is growing evidence to support the role of the homeobox transcription factors in normal hematopoietic differentiation as well as malignant hematopoiesis. The persistent, and inappropriate, expression of the homeobox gene HoxA9 has been described in the majority of acute myeloid leukemias. This implicates HoxA9 dysregulation as a common pathway of differentiation arrest in myeloid leukemias and suggests that by understanding and targeting this pathway, one might be able to overcome differentiation arrest. In cultures of primary murine bone marrow, constitutive expression of HoxA9 blocks myeloid differentiation and results in the outgrowth of immature myeloid cell lines. The mechanism by which HoxA9 causes differentiation arrest is not known and no compounds exist that inhibit HoxA9. We developed a murine cell line model in which the cells were blocked in differentiation by a conditional version of HoxA9. In this system, an estrogen-dependent ER-HoxA9 protein was generated by fusion with the estrogen receptor hormone-binding domain. When expressed in cultures of primary murine bone marrow, immortalized myeloblast cell lines can grow indefinitely in the presence of stem cell factor and beta-estradiol. Upon removal of beta-estradiol, and inactivation of HoxA9, these cell lines undergo synchronous and terminal myeloid differentiation. We took advantage of an available transgenic mouse model in which GFP was expressed downstream of the lysozyme promoter, a promoter expressed only in mature neutrophils and macrophages. Cell lines derived from the bone marrow of this lysozyme-GFP mouse were GFP-negative at baseline and brightly GFP-positive upon differentiation. In this manner, we generated a cell line with a built-in reporter of differentiation. These cells formed the basis of a high-throughput screen in which cells were incubated with small molecules for a period of four days in 384-well plate format. The cells were assayed by multi-parameter flow cytometry to assess for toxicity and differentiation. Compounds that triggered green fluorescence were scored as “HITS” and their pro-differentiation effects confirmed by analysis of morphology and cell surface markers. Given the availability of cells and the simple and reliable assay, we performed both a pilot screen of small molecules at The Broad Institute as well as an extensive screen of the NIH Molecular Libraries Small Molecule Repository. The screen of more than 350,000 small molecules was carried out in collaboration with the University of New Mexico Center for Molecular Discovery. We have identified one lead class of compounds - prostacyclin agonists – capable of promoting myeloid differentiation in this cell line model of AML. Using a parallel cell line derived from a prostacyclin receptor knock-out mouse, we confirmed that activity was due to signaling through the prostacyclin receptor. The role of prostacyclin signaling in myeloid differentiation has not been previously described. Analysis of gene expression demonstrated that the expression of the prostacyclin receptor is seen in ∼60% of in primary human AML samples. This is a potentially exciting finding as prostacyclin agonists (e.g. treprostinil) are clinically relevant as well as FDA-approved. Their potential role in the treatment of acute myeloid leukemia is unknown. Here we present the details of our high-throughput flow cytometry system and preliminary identification of pro-differentiation agents in AML. If successful, we anticipate that one of these small molecules may offer insight into a mechanism for overcoming differentiation arrest, and may also translate into a novel, clinically relevant treatment for acute myeloid leukemia. Disclosures: Sklar: IntelliCyt: Founder of IntelliCyt, the company that sells the HyperCyt high-throughput flow cytometry system. Other. Zon:Fate Therapeutics: Founder Other.

Autophagy Promotes the Survival and Therapy Resistance of Human Acute Myeloid Leukemia Cells Under Hypoxia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2236-2236 ◽  
Author(s):  
Dirkje W Hanekamp ◽  
Megan K Johnson ◽  
Scott Portwood ◽  
Joshua Acklin ◽  
Eunice S. Wang
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase Inhibitor ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Xenograft Models ◽  
Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy occurring primarily in older adults. Despite high remission rates following upfront therapy, the disease eventually recurs in most patients, and overall cure rates remain only 20-30%. Preclinical studies have recently demonstrated that the marrow microenvironment in acute leukemic hosts to be intrinsically hypoxic, with AML progression associated with further hypoxia. Moreover, human AML cells and primary AML colonies cultured under hypoxia are markedly less sensitive to cytarabine chemotherapy than normoxic cells. We hypothesized that AML cells may respond to hypoxic stress and mediate chemoresistance in part by invoking autophagy, a highly regulated catabolic process by which cells evade apoptosis by degrading damaged cellular components. To test our hypothesis, we investigated the effects of two known autophagy inhibitors (bafilomycin A1 (Baf) and chloroquine (CQ)) on the sensitivity of human AML cells to various therapeutic agents under differing oxygen levels. Methods: We treated HEL (FLT-3 wildtype) and MV4-11 (FLT-3 ITD mutant) AML cells with autophagy inhibitors (Baf and CQ) alone and in combination with a chemotherapeutic drug (cytarabine (AraC), doxorubicin (Dox), decitabine (Dac)) or a tyrosine kinase inhibitor (sorafenib, SFN) under normoxic (21% O2) or hypoxic (1% O2) conditions. Apoptosis /cell death and proliferation were measured by flow cytometry for Annexin-PI and MTT assays, respectively. Autophagy was assessed by flow cytometry using Cyto-ID Green Dye (Enzo Life Sciences), fluorescent microscropy for acridine orange dye accumulation, and western blot analysis. Results: Autophagy in human ALL and AML cell lines was significantly increased following 24-72 hours of hypoxia (1% O2) as compared with normoxia and was a relatively late response to prolonged low oxygen levels (> 24 hours). Treatment with cytotoxic agents (AraC or Dox) or hypomethylating agent (Dac) resulted in a dose-dependent increases in the number of autophagic vesicles in AML cells consistent with autophagy induction. Low-doses of Baf which selectively inhibits the vacuolar H+ ATPase to prevent lysosomal acidification, and CQ, which blocks lysosome-autophagosome fusion by raising the pH of lysosomes and endosomes, both resulted in buildup of autophagic vesicles by flow cytometry consistent with inhibition of autophagic flux in human AML cells. Combination treatment with an autophagy inhibitor (Baf, CQ) and cytotoxic chemotherapy (AraC, Dox) significantly enhanced apoptosis and cell death over single agent therapy. Treatment with Baf combined with hypomethylating therapy (Dac) synergistically improved the anti-leukemic effects as compared with monotherapy (CI 0.09-0.31)(see Figure). The addition of Baf also improved cell death induced by sorafenib (SFN) on FLT-3 ITD mutant human AML cells (MV4;11) (CI 0.36-0.9) (see Figure). Single agent Baf or CQ treatment resulted in significantly higher levels of apoptosis and cell death in AML cells under hypoxia. The anti-tumor activity of almost all combination regimens was consistently improved under hypoxic versus normoxic culture conditions. In vivo CQ treatment (25-50 mg/kg i.p. daily) in preclinical human AML xenograft models significantly inhibited systemic leukemia progression as a single agent. Further experiments investigating the in vivo effects of CQ combined with other chemotherapeutic agents in preclinical AML xenograft models are ongoing. Conclusions: Our data suggest that human AML cells preferentially induce autophagy to promote survival under chronic hypoxia and following cytotoxic, hypomethylating, and FLT-3 tyrosine kinase inhibitor therapy. Strategies targeting autophagy therefore may have the potential to improve therapeutic responses and overcome chemoresistance of AML cells within the hypoxic bone marrow microenvironment. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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