Dendrogenin_A : A Natural Liver X Receptor Modulator for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3767-3767
Author(s):  
Christian Recher ◽  
Marion David ◽  
Philippe de Medina ◽  
Cécile Bize ◽  
Nizar Serhan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Equity Ownership ◽  
Kg1 Cells ◽  
Acute Myeloid

Abstract Acute Myeloid Leukemia (AML) is the most common type of leukemia in adults. Despite intensive research, current treatments remain unsatisfactory with only 40% of younger (<60 years) and less than 10% of older (>60 years) AML patients achieving long-term complete remission. Consequently, drugs with novel mechanism of action are urgently needed to improve the outcome of these patients. We have recently identified Dendrogenin A (DDA) as a cholesterol metabolite present in normal cells but undetectable in various cancer cell lines including AML (de Medina et al, Nat Commun, 2013). DDA, the first steroidal alkaloid identified in mammals, exhibited strong anticancer effects against different tumor models in vitro and in vivo. In this study, we investigated the antileukemic potency of DDA in AML. We demonstrated that DDA exerts potent cytotoxic effect in a large panel of AML cell lines and cytogenetically and molecularly diverse primary AML patient samples (n=50) with a median IC50 of 3.3 µM (range 1.2-10 µM). We determined that DDA triggers both apoptosis and cytotoxic autophagy on AML cells. Macroautophagy was characterized by the accumulation of autophagic vacuoles and the stimulation of autophagic flux. As opposed to conventional chemotherapies, the antileukemic effect of DDA was similarly efficient in both immature stem/progenitor CD34+CD38-CD123+ subpopulation and leukemic bulk. Interestingly, the antileukemic activity of DDA on AML patient samples was not correlated to usual prognostic factors such as adverse cytogenetic risk karyotype, clonogenic ability, white blood cells count and FLT3-ITD or NPM status. Pharmacokinetic studies revealed that both per os (PO) and intraperitoneal (IP) administration led to a good absorption with calculated bioavailability of 74% (PO) and 48% (IP), showing that these modes of administration are relevant to in vivo preclinical studies. We then examined the in vivo anti-leukemic efficacy of DDA in NOD/SCID mice injected subcutaneously with HL60 and KG1 cells. We demonstrated that daily administration of DDA (20 mg/kg IP or 40 mg/kg PO) significantly reduced KG1 and HL60 tumor growth. Immunohistochemical analysis revealed that AML xenografts from mice exposed to DDA display a 3.5 fold increase of LC3 punctated cells and a decreased P62 level highlighting that DDA induces autophagy in vivo. Furthermore, DDA significantly kills AML cells in bone marrow and brain (55±5.6% reduction of viable CD45+ cells), and strongly reduces (57±7.8%) the total cell tumor burden in bone marrow and spleen in established disease models (eg. orthotopically engraftment of HL60 cells and three primary AML patient cells via tail vein injection in NOD/SCID/IL2Rγc-deficient mice). In addition, we showed that DDA is well tolerated in mice at effective dose and spares normal hematopoietic stem/progenitor cells from healthy donor. Mechanistic studies revealed that DDA is a natural modulator of the Liver X Receptor (LXR), a nuclear receptor involved in cholesterol homeostasis, immunity and proliferation. We found that the silencing of LXRβ gene prevents the capacity of DDA to trigger both cell death and autophagy on AML cells in vitro. In addition, DDA failed to block tumor development and to trigger autophagy on LXRβ-invalidated KG1 cells xenografted on NOD/SCID mice. Moreover, DDA strongly stimulates the expression of the myeloid leukemogenesis tumor suppressors Nur77 and Nor1 through an LXRβ-dependent mechanism. Interestingly, DDA triggers the relocation of Nur77 to the mitochondria, a process associated with both apoptosis and autophagic cell death. This study provides a strong rationale to bring DDA in clinical trials for patients with AML. Disclosures de Medina: Affichem: Employment. Bize:Affichem: Employment. Paillasse:Affichem: Employment. Noguer:Affichem: Employment. Sarry:Affichem: Equity Ownership. Silvente-Poirot:Affichem: Equity Ownership. Poirot:Affichem: Equity Ownership.

The PI3K Inhibitor GDC-0941 Induces Growth Arrest and Apoptosis of Acute Myeloid Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3787-3787
Author(s):  
Laura Sun ◽  
Leanne Berry ◽  
Changchun Du ◽  
Xiaoju Max Ma ◽  
Lily Shi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Pi3k Inhibitor ◽  
Employment Equity ◽  
Disease Heterogeneity ◽  
Equity Ownership ◽  
Acute Myeloid

Abstract Abstract 3787 Poster Board III-723 Acute myeloid leukemia (AML) is a biologically and molecularly heterogeneous malignancy characterized by an accumulation of myeloid-lineage cells in the blood and bone marrow. Phosphatidylinositol 3' kinase (PI3K) -mediated signaling is frequently dysregulated in cancer and controls fundamental cellular functions such as cell migration, growth, survival and development of drug resistance in many cancers, including AML, and therefore represents an attractive therapeutic target, even against the backdrop of AML disease heterogeneity. In AML, for example, activating mutations in Ras and Flt3 are common and a number of autocrine and paracrine signaling mechanisms have been proposed as important disease mediators; all converge on PI3K as an important signal transduction node to provide growth- and survival-promoting effects. Here, we demonstrate that PI3K p110-a,b,d catalytic subunits are all prevalent across a panel of ∼30 cell lines and that the majority of cell lines as well as primary patient samples show evidence of PI3K pathway activation. We demonstrate in vitro with both cell lines and patient cell isolates, that a potent and selective pan-isoform PI3K inhibitor, GDC-0941, modulates pharmacodynamic markers such as p-Akt, p-4E-BP1, and p-FOXO-3a, and that treated cells show a G0/G1 cell-cycle arrest. A strong induction of apoptosis is observed by annexin/PI staining with upregulation of cleaved caspases and PARP. Preliminary observations suggest significant induction of p27 and Bim as direct transcriptional targets of FOXO-3a, as well as downregulation of c-Myc levels; these targets may represent key mediators of observed cellular effects and are the subject of ongoing work. Additional studies are currently underway to identify clinical standard-of-care agents and new molecular entities (pipeline NMEs) that combine favorably with PI3k-inhibition in vitro, and the results of several in-vivo xenograft studies will also be presented. Disclosures: Sun: Genentech Inc.: Employment. Berry:Genentech: Employment, Patents & Royalties. Du:Genentech, Inc.: Employment, Equity Ownership. Ma:Genentech: Employment, Patents & Royalties. Shi:Genentech: Employment, Patents & Royalties. Medeiros:Genentech: Research Funding. Ebens:Genentech, Inc.: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Potentially Curative Therapeutic Activity of NEO212, a Perillyl Alcohol-Temozolomide Conjugate, in Preclinical Cytarabine-Resistant Models of Acute Myeloid Leukemia

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3385
Author(s):  
Axel H. Schönthal ◽  
Steve Swenson ◽  
Radu O. Minea ◽  
Hye Na Kim ◽  
Heeyeon Cho ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Side Effects ◽  
Anticancer Activity ◽  
Myeloid Leukemia ◽  
Perillyl Alcohol ◽  
Therapeutic Activity ◽  
Acute Myeloid

Despite progress in the treatment of acute myeloid leukemia (AML), the clinical outcome remains suboptimal and many patients are still dying from this disease. First-line treatment consists of chemotherapy, which typically includes cytarabine (AraC), either alone or in combination with anthracyclines, but drug resistance can develop and significantly worsen prognosis. Better treatments are needed. We are developing a novel anticancer compound, NEO212, that was created by covalent conjugation of two different molecules with already established anticancer activity, the alkylating agent temozolomide (TMZ) and the natural monoterpene perillyl alcohol (POH). We investigated the anticancer activity of NEO212 in several in vitro and in vivo models of AML. Human HL60 and U937 AML cell lines, as well as different AraC-resistant AML cell lines, were treated with NEO212 and effects on cell proliferation, cell cycle, and cell death were investigated. Mice with implanted AraC-sensitive or AraC-resistant AML cells were dosed with oral NEO212, and animal survival was monitored. Our in vitro experiments show that treatment of cells with NEO212 results in growth inhibition via potent G2 arrest, which is followed by apoptotic cell death. Intriguingly, NEO212 was equally potent in highly AraC-resistant cells. In vivo, NEO212 treatment strikingly extended survival of AML mice and the majority of treated mice continued to thrive and survive without any signs of illness. At the same time, we were unable to detect toxic side effects of NEO212 treatment. All in all, the absence of side effects, combined with striking therapeutic activity even in an AraC-resistant context, suggests that NEO212 should be developed further toward clinical testing.

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.

The BTK Inhibitor Ibrutinib Blocks SDF1/CXCR4 Mediated Migration of Acute Myeloid Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 915-915
Author(s):  
Stuart A Rushworth ◽  
Lyubov Zaitseva ◽  
Megan Y Murray ◽  
Matthew J Lawes ◽  
David J MacEwan ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Cell Trafficking ◽  
Acute Myeloid

Abstract Introduction Despite recent significant progress in the understanding of the biology of acute myeloid leukemia (AML) the clinical outcomes for the majority of patients diagnosed with AML presently remain poor. Consequently, there is an urgent need to identify pharmacological strategies in AML, which are not only effective but can be tolerated by the older, less well patient. Recently our group and others have shown that there is high Bruton’s Tyrosine Kinase (BTK) phosphorylation and RNA expression in AML. Moreover, our recent study described for the first time that ibrutinib and BTK-targeted RNA interference reduced factor-induced proliferation of both AML cell lines and primary AML blasts, as well as reducing AML blast adhesion to bone marrow stromal cells. Inhibition of BTK has been shown to regulate chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma cell migration by inhibiting SDF1 (stromal derived factor 1) induced CXCR4 regulated cell trafficking. Here we report that in human AML ibrutinib in addition functions in a similar way to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable in vivo. Methods To investigate the role of BTK in regulating AML migration we used both pharmacological inhibitor ibrutinib and genetic knockdown using a lentivirus mediated BTK targeted miRNA in primary AML blasts and AML cell lines. We examined migration of AML blasts and AML cells to SDF-1 using Transwell permeable plates with 8.0µM pores. Western blotting was used to examine the role of SDF-1 in regulating BTK, AKT and MAPK activation in primary AML blasts. Results We initially examined the expression of CXCR4 in human AML cell lines and found that 4/4 cell lines were positive for CXCR4 expression. Next we examined the effects of ibrutinib on the migration of the AML cell lines U937, MV4-11, HL60 and THP-1 in response to SDF1. We found that ibrutinib can inhibit the migration of all AML cell lines tested. We tested the in-vitro activity of ibrutinib on SDF-1 induced migration in a spectrum of primary AML blasts from a wide age spectrum of adult patients and across a range of WHO AML subclasses and found that ibrutinib significantly inhibits primary AML blast migration (n=12). Next we found that ibrutinib can inhibit SDF-1 induced BTK phosphorylation and downstream MAPK and AKT signalling in primary AML blast. Finally to eliminate the problems associated with off target ibrutinib activity we evaluated migration of AML cells lines using genetic inhibition of BTK. The introduction of BTK-specific miRNA dramatically inhibited the expression of BTK in THP-1 and HL60 and reduced SDF1 mediated migration confirming that BTK is involved in regulating AML migration in response to SDF1. Conclusions These results reported here provide a molecular mechanistic rationale for clinically evaluating BTK inhibition in AML patients and suggests that in some AML patients the blasts count may initially rise in response to ibrutinib therapy, analgous to similar clinical observations in CLL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibiting Mitochondrial Complex II Exposes a Novel Metabolic Vulnerability in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1300-1300
Author(s):  
Alessia Roma ◽  
Matthew Tcheng ◽  
Nawaz Ahmed ◽  
Sarah Walker ◽  
Preethi Jayanth ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Succinate Dehydrogenase ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Tca Cycle ◽  
Cell Fates ◽  
Reductive Carboxylation ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a hematological malignancy, characterized by an increased reliance on mitochondria-related energetic pathways including oxidative phosphorylation (OXPHOS). Consistent with this, the electron transport chain (ETC), a component of OXPHOS has been demonstrated to be a suitable anti-leukemia target, with ETC complex I inhibitors currently in clinical development. Relative to its counterparts, complex II (CII) is unique in that it directly links the ETC to the tricarboxylic acid (TCA) cycle through succinate dehydrogenase (SDH) activity. Moreover, it is the only ETC complex with elevated activity in AML, relative to normal hematopoietic samples, with indirect inhibition selectively targeting AML cells. However, direct CII inhibition in AML has not been previously investigated, nor have the mechanisms underlying the divergent fates of AML and normal cells upon CII inhibition. A genetic approach was first used to assess the effects of CII impairment on AML growth in vitro and in vivo. Using lentiviral mediated shRNA we generated AML cell lines lacking succinate dehydrogenase assembly factor 1 (Sdhaf1). Sdhaf1 knockdown suppressed CII activity, cell proliferation and clonogenic growth across all three cell lines and delayed leukemia growth in vivo. To recapitulate these effects through a pharmacological approach, we aimed to identify a novel CII inhibitor, since currently available inhibitors are only effective at high doses and are neurotoxic. Through an in silico structural screen and molecular docking study, shikonin was identified as a small molecule that selectively binds to CII. Shikonin inhibited CII activity in the AML cells lines and patient-derived samples, and selectively killed AML cells (EC 50: 1.0μM ± 0.04) while sparing normal progenitors. In murine engraftment models, shikonin (2.0-3.0 mg/kg, 3x/week for 5 weeks) significantly reduced engraftment of patient-derived AML cells but had no effect on normal hematopoiesis. To further characterize the mechanisms governing the divergent cell fates of CII inhibition, we performed stable isotope metabolic tracing using 13C 6- glucose and 13C 5, 15N 2-glutamine in patient-derived AML cells and normal mobilized peripheral blood mononuclear cells (MNCs). Both pharmacological and genetic loss of CII resulted in TCA cycle truncation by impairing oxidative metabolism of both glucose and glutamine. In Sdhaf1 knockdown and primary AML cells, this led to a depletion in steady state levels of TCA metabolites proceeding SDH. Inhibition of CII most notably suppressed levels of aspartate, a nucleotide precursor whose levels dictate the proliferative capacity of a cell under ETC dysfunction. Remarkably, MNCs maintained aspartate levels despite inhibition of CII, which was attributed to reductive carboxylation of glutamine, an alternate metabolic pathway that can regenerate TCA intermediates when OXPHOS is impaired. In contrast, while reductive carboxylation was also active in AML cells after CII inhibition, this activity was insufficient to maintain aspartate levels and resulted in metabolite depletion and cell death. Thus, loss of CII activity results in diverse cell fates whereby normal haematopoietic, but not AML cells sufficiently use reductive carboxylation of glutamine to overcome TCA cycle truncation, sustain aspartate levels and avert cell death. This is further evident through modulation of glutamine entry into the TCA cycle, where supplementation of cell-permeable α-ketoglutarate abrogated shikonin-mediated cell death while concomitant treatment with the glutaminase inhibitor CB-839, sensitized cells. Together, these results expose reductive carboxylation to support aspartate biosynthesis, as a novel metabolic vulnerability in AML that can be pharmacologically targeted through CII inhibition for clinical benefit. Disclosures Minden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

scholarly journals The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1663 ◽  
Author(s):  
Arne Velthaus ◽  
Kerstin Cornils ◽  
Jan K. Hennigs ◽  
Saskia Grüb ◽  
Hauke Stamm ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Binding Protein ◽  
Actin Binding ◽  
Bone Marrow Niche ◽  
Actin Binding Protein ◽  
Acute Myeloid

Leukemia-initiating cells reside within the bone marrow in specialized niches where they undergo complex interactions with their surrounding stromal cells. We have identified the actin-binding protein Plastin-3 (PLS3) as potential player within the leukemic bone marrow niche and investigated its functional role in acute myeloid leukemia. High expression of PLS3 was associated with a poor overall and event-free survival for AML patients. These findings were supported by functional in vitro and in vivo experiments. AML cells with a PLS3 knockdown showed significantly reduced colony numbers in vitro while the PLS3 overexpression variants resulted in significantly enhanced colony numbers compared to their respective controls. Furthermore, the survival of NSG mice transplanted with the PLS3 knockdown cells showed a significantly prolonged survival in comparison to mice transplanted with the control AML cells. Further studies should focus on the underlying leukemia-promoting mechanisms and investigate PLS3 as therapeutic target.

Anti-Acute Myeloid Leukemia Activity of Chaetocin, a Novel Epigenetic Drug Inhibitor Inducing Oxidative Stress.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 889-889
Author(s):  
Hassiba Chaib ◽  
Thomas Prebet ◽  
Audrey Restouin ◽  
Remy Castellano ◽  
Sandrine Opi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Hdac Inhibitors ◽  
Oxidative Species ◽  
Acute Myeloid

Abstract Recent studies have highlighted the importance of epigenetic modifications in the pathogenesis of Acute Myeloid Leukemia (AML). This results have been confirmed by the activity of new drug like DNA demethylating agents and histone deacetylase (HDAC) inhibitors in both in vivo and in vitro studies. Recently, Chaetocin, a natural fungal compound, has been identified as the first specific inhibitor of the histone methyltransferase SU(VAR)3–9 which plays a role in heterochromatin gene silencing. In this study, we decided to evaluate Chaetocin as a therapeutic agent in AML in vitro and to explore the related mechanisms. We show that Chaetocin induce dramatic cell death at nanomolar concentrations in U937 and HL60 (97.2% ± 0.4 and 91.6% ± 9 cell death at 100 nM chaetocin, respectively), and to a lesser extend in K562 (67.3% ± 1.6 cell death at 100 nM chaetocin), cell cultures. Cell death occurred at 24 h incubation time which correlated with induction of apoptosis as assessed by Annexin V/7-AAD staining and activation of downstream executioner caspase-3/7. Using transcription low-density array and quantitative RT- PCR, Chaetocin was showed to up-regulate gene transcription such as of the cell cycle inhibitor p21/WAF1 consistent with a role for the targeted SU(VAR)3–9 in heterochromatin gene silencing. In agreement with the recent report of Chaetocin being a promising new antimyeloma agent acting via imposition of oxidative stress, intracellular levels of oxidative species were increased in Chaetocin treated U937 cells in a time- and dose-dependent manner that correlated with induction of cell death. Furthermore, incubation of cells with N-acetyl cysteine, a cell-permeable precursor of intracellular glutathione reductant, prevented chaetocin-induced accumulation of oxidative species, transcription of selected genes (e.g. p21/WAF1), activation of caspase-3, and cell death. Finally, Chaetocin was found to increase the antileukemia activity of HDAC inhibitors and Aracytin, and thus appears as a promising agent for further study as a potential anti-AML therapeutic. Preliminary results obtained in vivo in xenograft models and ex vivo, using blasts of a panel of patients with AML, will be presented.

The Novel Fluoropyrimidine FdUMP[10] Is Highly Active Against Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3302-3302
Author(s):  
Timothy Pardee ◽  
Evan Gomes ◽  
Jamie Jennings-Gee ◽  
David L. Caudell ◽  
William Gmeiner
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Single Agent ◽  
The Novel ◽  
Highly Active ◽  
Ic50 Values ◽  
Acute Myeloid

Abstract Abstract 3302 Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy that leads to marrow failure and death. This disease affects approximately 12,000 people per year in the United States, causing 9,000 deaths. Despite decades of research, therapy remains essentially unchanged and outcomes are poor. In patients over the age of 60 less then 10% of patients survive 5 years from diagnosis. There is a desperate need for the identification of new active agents with favorable toxicity profiles. The novel polymeric fluoropyrimidine (FP) FdUMP[10] is an oligodeoxynucleotide pro-drug of the thymidylate synthase (TS)-inhibitory FP metabolite 5-fluoro-2'-deoxyuridine-5`-O-monophosphate (FdUMP). The observation that this compound was highly active against several leukemia lines in the NCI 60 cell line screen prompted us to evaluate its activity in several preclinical models of AML. In vitro, FdUMP[10] exhibited remarkable activity against 3 human acute leukemia cell lines, HL60, Jurkat and THP-1, with IC50 values of 3.378 nM (95% CI 2.984 to 3.825), 5.438 nM (4.609 to 6.417) and 4.093 nM (3.413 to 4.907) respectively. We next tested its efficacy against a more genetically defined murine model of AML driven by expression of MLL-ENL. FdUMP[10] exhibited even greater activity against all murine lines tested. The IC50 values of FdUMP[10] against two MLL-ENL driven murine AML cell lines were 214 pM (95%CI 178.9 to 255.9) and 292.3 pM (251.8 to 339.4). The IC50 values observed for FdUMP[10] for all the murine lines tested were lower than both Ara-C (30-40 nM) and doxorubicin (2-4 nM). We then determined the cytotoxic mechanism for FdUMP[10] in vitro. Upon treatment with FdUMP[10] both the human and murine cell lines undergo extensive apoptosis as indicated by Annexin V and propidium iodide staining. Treated cells developed γH2AX foci, rapid and complete TS inhibition and display trapped Topoisomerase I (Topo I) cleavage complexes. FdUMP[10]-mediated induction of apoptosis was p53 independent as murine AML cells that had p53 knocked down by RNAi demonstrated resistance to both Ara-C and doxorubicin, but not to FdUMP[10]. We next tested the efficacy of FdUMP[10] in vivo. The MLL-ENL driven murine AML model results in blasts that can be transplanted into sublethally irradiated, immunocompetent, syngeneic recipients. The recipients develop a fatal and therapy-resistant AML. Lines were generated that expressed a luciferase reporter. Animals were imaged 6–7 days after injection of the leukemias to ensure engraftment and then began treatment with either the combination of Ara-C plus doxorubicin, single-agent FdUMP[10], or observation. Studies were performed using 2 doses of FdUMP[10] at 150 or 300 mg/kg injected on days 1 and 3 and compared to animals treated with 100 mg/kg Ara-C and 3mg/kg doxorubicin injected on days 1 through 5. Both treatments resulted in a statistically significant survival advantage over observation. A preliminary toxicology study compared FdUMP[10], 150 mg/kg daily, to 5-fluorouracil (5 FU), 150 mg/kg daily, or the combination of Ara-C at 100 mg/kg plus doxorubicin at 3 mg/kg daily. All groups were treated for 3, 4 or 5 days. On day 6 animals were sacrificed and organs harvested, sectioned, and stained. Slides were then reviewed by a veterinary pathologist. Tissues most affected were the small intestine, colon, and the bone marrow. The 5FU-treated animals had severe villous blunting and fusion with crypt necrosis in both large and small intestine. In contrast, FdUMP[10]-treated animals had only mild crypt epithelial apoptosis with mitoses. The 5 FU and Ara-C plus doxorubicin groups had a severe pan-cytopenia in the marrow compared to FdUMP[10] treated animals that showed only minimal to mild apoptosis. These data support the assertion that FdUMP[10] has lower toxicity then either Ara-C plus doxorubicin or identically dosed 5 FU. In summary FdUMP[10] exhibited remarkable activity against AML cells in vitro and in vivo. Additionally, FdUMP[10] had decreased toxicity compared to treatment with either single agent 5 FU or combination treatment with Ara-C plus doxorubicin. Disclosures: Gmeiner: Salzburg Therapeutics: Equity Ownership.

Expression of Bcl-2 Pro-Survival Family Proteins Predicts Pharmacological Responses to ABT-199, a Novel and Selective Bcl-2 Antagonist, in Multiple Myeloma Models

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5028-5028 ◽  
Author(s):  
Deepak Sampath ◽  
Elizabeth Punnoose ◽  
Erwin R. Boghaert ◽  
Lisa Belmont ◽  
Jun Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Cell Lines ◽  
Single Agent ◽  
Employment Equity ◽  
Bone Marrow Biopsies ◽  
Xenograft Models ◽  
Equity Ownership

Abstract Abstract 5028 Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM. A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity. ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures: Sampath: Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

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