Pre-Clinical Combination of AO-176, a Highly Differentiated Clinical Stage CD47 Antibody, with Either Azacitidine or Venetoclax Significantly Enhances DAMP Induction and Phagocytosis of Acute Myeloid Leukemia

While T cell checkpoint inhibitors are mainstays of cancer immunotherapy, therapies that direct innate immune responses against cancer are lacking. CD47, a "don't eat me" signal, is an innate immune cell checkpoint which binds SIRPα on macrophages and dendritic cells to limit phagocytosis and its upregulation on tumor cells leads to evasion of immune detection and clearance. Therapeutic antibodies have previously been developed to block CD47 and induce phagocytosis of tumor cells, thus validating the pathway. AO-176, a next generation humanized IgG2 anti-CD47 antibody, was developed to block the CD47/SIRPα interaction and induce tumor cell phagocytosis. Moreover, AO-176 directly kills tumor cells through a non-ADCC-dependent mechanism via induction of programmed cell death type III. In addition to these tumor eliminating properties, AO-176 has the potential for a strong safety profile as a result of its preferential binding to tumor versus normal cells, lack of RBC binding, and enhanced binding to tumor cells at acidic pH. CD47 has previously been shown to be upregulated on acute myeloid leukemia (AML) leukemic stem cells (LSC), enabling their expansion through evasion from phagocytic clearance. As a result, patients with increased CD47 on AML LSCs have worse overall survival. In this study, AO-176 efficacy was evaluated in AML cell lines as a single agent and in combination with azacitidine and venetoclax which are approved therapies for AML. Azacitidine is a cytosine analogue which acts to inhibit DNA methylation, and venetoclax is a potent Bcl-2 inhibitor. Previous studies have shown that azacitidine induces apoptosis of tumor cells and increases cell surface exposure of calreticulin, a DAMP (Damage Associate Molecular Pattern) which provides a strong pro-phagocytic signal. From these findings, it was hypothesized that azacitidine would enable increased tumor cell phagocytosis when combined with AO-176. The potential for a similar enhancement with a combination of AO-176 and venetoclax was also explored. The ability of AO-176, with or without azacitidine or venetoclax, to induce DAMPs on the surface of AML cells was assessed. Cell surface expression of DAMPs, calreticulin and PDIA3, were measured by flow cytometry. AO-176, azacitidine, and venetoclax as single agents potently increased both calreticulin and PDIA3 in a dose-dependent manner on AML cell lines such as HL60 This is the first time, to our knowledge, that venetoclax has been shown to induce DAMPs. To better understand the functional implications of these findings, in vitro phagocytosis assays were performed. Azacitidine and venetoclax significantly enhanced AO-176-mediated phagocytosis of AML cells compared to any of the agents alone. Moreover, when AO-176 was combined with azacitidine in direct tumor cell killing assays, enhanced activity was observed in a subset of AML cell lines. In conclusion, AO-176 combined with either azacitidine or venetoclax, resulted in significant enhancement of phagocytic AML cell clearance in vitro which also correlated with the ability of these agents to induce DAMPs. In vivo treatment with AO-176 in combination with these agents is in progress. AO-176 is being evaluated in phase 1 clinical trials for the treatment of patients with solid tumors (NCT03834948) and multiple myeloma (NCT04445701). Disclosures Donio: Arch Oncology: Current Employment, Current equity holder in private company. Wilson:Arch Oncology: Current Employment, Current equity holder in private company. Darwech:Arch Oncology: Current Employment, Current equity holder in private company. Andrejeva:Arch Oncology: Current Employment, Current equity holder in private company. Capoccia:Arch Oncology: Current Employment, Current equity holder in private company. Puro:Arch Oncology: Current Employment, Current equity holder in private company. Kashyap:Arch Oncology: Current Employment, Current equity holder in private company. Pereira:Arch Oncology: Current Employment, Current equity holder in private company.

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

Archives of Medical Science ◽

10.5114/aoms/142465 ◽

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.

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

10.1101/636969 ◽

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.

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The Vitamin E Derivative Gamma Tocotrienol Promotes Anti-Tumor Effects in Acute Myeloid Leukemia Cell Lines

Nutrients ◽

10.3390/nu11112808 ◽

2019 ◽

Vol 11 (11) ◽

pp. 2808 ◽

Cited By ~ 6

Author(s):

Ghanem ◽

Zouein ◽

Mohamad ◽

Hodroj ◽

Haykal ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Vitamin E ◽

Cell Lines ◽

Myeloid Leukemia ◽

Apoptotic Cell ◽

Leukemia Cell ◽

Therapeutic Effects ◽

Apoptotic Pathway ◽

Acute Myeloid

Acute myeloid leukemia (AML) is a blood cancer characterized by the formation of faulty defective myelogenous cells with morphological heterogeneity and cytogenic aberrations leading to a loss of their function. In an attempt to find an effective and safe AML treatment, vitamin E derivatives, including tocopherols were considered as potential anti-tumor compounds. Recently, other isoforms of vitamin E, namely tocotrienols have been proposed as potential potent anti-cancerous agents, displaying promising therapeutic effects in different cancer types. In this study we evaluated the anti-cancerous effects of γ-tocotrienol, on AML cell lines in vitro. For this purpose, AML cell lines incubated with γ-tocotrienol were examined for their viability, cell cycle status, apoptotic cell death, DNA fragmentation, production of reactive oxygen species and expression of proapoptotic proteins. Our results showed that γ-tocotrienol exhibits time and dose-dependent anti-proliferative, pro-apoptotic and antioxidant effects on U937 and KG-1 cell lines, through the upregulation of proteins involved in the intrinsic apoptotic pathway.

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Structure-Based Drug Design of c-Kit Inhibitors for Use in the Treatment of Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.1906.1906 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1906-1906

Author(s):

David S. Maxwell ◽

Ashutosh Pal ◽

Zhenghong Peng ◽

Alexandr Shavrin ◽

Stefan Faderl ◽

...

Keyword(s):

Crystal Structure ◽

Acute Myeloid Leukemia ◽

Drug Design ◽

Cell Lines ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Kinase Assay ◽

Structure Based Drug Design ◽

Acute Myeloid

Abstract Inhibitors of c-Kit kinase have shown clinical relevance in various myeloid disorders, including acute myeloid leukemia (AML). Research in our lab has been oriented towards structure-based drug design of c-Kit inhibitors based on the available crystal structure. We describe the design, synthesis, and preliminary results from the in-vitro testing of several c-Kit kinase inhibitors in both enzymatic and cell-based assays. The design resulted from in-silico screening of several targeted libraries via docking to the crystal structure of c-Kit, followed by aggressive post-filtering by several criteria to significantly bias synthesis efforts towards candidate compounds with best chance for success. This led to 128 structures built from 8 common structural cores, from which 2 cores were initially selected based on the synthetic feasibility. Five compounds were initially synthesized, and were immediately followed by 60 compounds with variations to probe local structure-activity relationships. The initial set of compounds, designated APCKxxx, was tested in a c-Kit kinase assay; two compounds were found to have an IC50 in the high nM to low uM range. These compounds have been tested in a MTT-based assay using OCIM2 and OCI/AML3 cell lines. In the c-Kit expressing OCI/AML3 cell line, all five compounds possessed an EC50 < 500 nM and two had and EC50 ~100 nM. Our most recent results show that these compounds also show efficacy in some imatinib-resistant cell lines. We will discuss these results and our strategies for the second generation of compounds that are optimized for better activity, selectivity, and ADME properties.

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Aberrant Expression of the Homeobox Gene CDX2 in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.8.8 ◽

2006 ◽

Vol 108 (11) ◽

pp. 8-8 ◽

Cited By ~ 1

Author(s):

Claudia Scholl ◽

Dimple Bansal ◽

Konstanze Dohner ◽

Karina Eiwen ◽

Benjamin H. Lee ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Gene Copy ◽

Coding Region ◽

Aberrant Expression ◽

Cdx2 Expression ◽

Frequent Event ◽

Acute Myeloid

Abstract The caudal-type homeobox transcription factor 2 (CDX2) plays an important role in embryonic development and regulates the proliferation and differentiation of intestinal epithelial cells in the adult. Ectopic expression of CDX2 in the hematopoietic compartment was previously identified as the key pathogenetic event in a single patient with acute myeloid leukemia (AML) and t(12;13)(p13;q12). Using real-time quantitative PCR, we detected aberrant CDX2 expression in 153 (90%) of 170 patients with AML, in patients with high-risk myelodysplastic syndrome or advanced-stage chronic myeloid leukemia, and in several AML cell lines, but not in bone marrow derived from normal individuals. Expression of CDX2 was monoallelic in the majority of cases with informative single-nucleotide polymorphisms in the CDX2 coding region, but was not related to mutations in the CDX2 coding region or in the predicted CDX2 promoter sequence, gene-specific hypomethylation of the CDX2 promoter, or increased CDX2 gene copy numbers. Stable knockdown of CDX2 expression by lentivirus-mediated RNA interference inhibited the proliferation of various human AML cell lines exhibiting CDX2 transcript levels that were in the range of those observed in most primary AML samples, and strongly reduced their clonogenic potential in vitro. Primary murine hematopoietic progenitor cells transduced with Cdx2 acquired serial replating activity in vitro, could be continuously propagated in liquid culture, generated a fully penetrant and transplantable AML in vivo, and displayed dysregulated expression of Hox family members. Together, these results (i) demonstrate that aberrant expression of CDX2 is a frequent event in myeloid leukemogenesis, (ii) suggest a role for CDX2 as part of a common effector pathway that increases the proliferative capacity and promotes the self-renewal potential of hematopoietic progenitors, and (iii) support the unifying hypothesis that CDX2 is responsible, at least in part, for abnormalities in HOX gene expression in AML.

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Deregulated Apoptotic Pathways Point to Effectiveness of IAP Inhibitor Therapy in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.1275.1275 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1275-1275

Author(s):

Sonja C Lück ◽

Annika C Russ ◽

Konstanze Döhner ◽

Ursula Botzenhardt ◽

Domagoj Vucic ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Gene Set Enrichment Analysis ◽

Core Binding Factor ◽

Binding Factor ◽

Acute Myeloid

Abstract Abstract 1275 Poster Board I-297 Core binding factor (CBF) leukemias, characterized by translocations t(8;21) or inv(16)/t(16;16) targeting the core binding factor, constitute acute myeloid leukemia (AML) subgroups with favorable prognosis. However, 40-50% of patients relapse, and the current classification system does not fully reflect the heterogeneity existing within the cytogenetic subgroups. Therefore, illuminating the biological mechanisms underlying these differences is important for an optimization of therapy. Previously, gene expression profiling (GEP) revealed two distinct CBF leukemia subgroups displaying significant outcome differences (Bullinger et al., Blood 2007). In order to further characterize these GEP defined CBF subgroups, we again used gene expression profiles to identify cell line models similar to the respective CBF cohorts. Treatment of these cell lines with cytarabine (araC) revealed a differential response to the drug as expected based on the expression patterns reflecting the CBF subgroups. In accordance, the cell lines resembling the inferior outcome CBF cohort (ME-1, MONO-MAC-1, OCI-AML2) were less sensitive to araC than those modeling the good prognostic subgroup (Kasumi-1, HEL, MV4-11). A previous gene set enrichment analysis had identified the pathways Caspase cascade in apoptosis and Role of mitochondria in apoptotic signaling among the most significant differentially regulated BioCarta pathways distinguishing the two CBF leukemia subgroups. Thus, we concluded that those pathways might be interesting targets for specific intervention, as deregulated apoptosis underlying the distinct subgroups should also result in a subgroup specific sensitivity to apoptotic stimuli. Therefore, we treated our model cell lines with the Smac mimetic BV6, which antagonizes inhibitor of apoptosis (IAP) proteins that are differentially expressed among our CBF cohorts. In general, sensitivity to BV6 treatment was higher in the cell lines corresponding to the subgroup with good outcome. Time-course experiments with the CBF leukemia cell line Kasumi-1 suggested a role for caspases in this response. Interestingly, combination treatment of araC and BV6 in Kasumi-1 showed a synergistic effect of these drugs, with the underlying mechanisms being currently further investigated. Based on the promising sensitivity to BV6 treatment in some cell lines, we next treated mononuclear cells (mostly leukemic blasts) derived from newly diagnosed AML patients with BV6 in vitro to evaluate BV6 potency in primary leukemia samples. Interestingly, in vitro BV6 treatment also discriminated AML cases into two distinct populations. Most patient samples were sensitive to BV6 monotherapy, but about one-third of cases were resistant even at higher BV6 dosage. GEP of BV6 sensitive patients (at 24h following either BV6 or DMSO treatment) provided insights into BV6-induced pathway alterations in the primary AML patient samples, which included apoptosis-related pathways. In contrast to the BV6 sensitive patients, GEP analyses of BV6 resistant cases revealed no differential regulation of apoptosis-related pathways in this cohort. These results provide evidence that targeting deregulated apoptosis pathways by Smac mimetics might represent a promising new therapeutic approach in AML and that GEP might be used to predict response to therapy, thereby enabling novel individual risk-adapted therapeutic approaches. Disclosures Vucic: Genentech, Inc.: Employment. Deshayes:Genentech, Inc.: Employment.

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Inhibition Of LSD1 As a Therapeutic Strategy For The Treatment Of Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.3964.3964 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3964-3964 ◽

Cited By ~ 14

Author(s):

Ryan G. Kruger ◽

Helai Mohammad ◽

Kimberly Smitheman ◽

Monica Cusan ◽

Yan Liu ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Cell Surface ◽

Growth Inhibition ◽

Cell Lines ◽

Myeloid Leukemia ◽

Stem Cell Differentiation ◽

Ethical Review Process ◽

Acute Myeloid

Abstract Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. These complexes include Histone Deacetylases (HDAC1/2) and Co-Repressor for Element-1-Silencing Transcription factor (CoREST). LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression. LSD1 has been shown to play a role in development in various contexts. LSD1 can interact with pluripotency factors in human embryonic stem cells and is important for decommissioning enhancers in stem cell differentiation. Beyond embryonic settings, LSD1 is also critical for hematopoietic differentiation. LSD1 is overexpressed in multiple cancer types and recent studies suggest inhibition of LSD1 reactivates the all-trans retinoic acid receptor pathway in acute myeloid leukemia (AML). These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and through its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel LSD1 inhibitor (GSK2879552) in AML. GSK2879552 is a potent, selective, mechanism-based, irreversible inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML cells have a unique requirement for LSD1. While LSD1 inhibition did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of putative LSD1 target genes. This increase in the transcriptionally activating histone modification correlated with a dose dependent increase in gene expression. Treatment with GSK2879552 promoted the expression of cell surface markers, including CD11b and CD86, associated with a differentiated immunophenotype in 12 of 13 AML cell lines. For example, in SKM-1 cells, increases in cell surface expression of CD86 and CD11b occurred after as early as one day of treatment with EC50 values of 13 and 7 nM respectively. In a separate study using an MV-4-11 engraftment model, increases in CD86 and CD11b were observed as early as 8 hours post dosing. GSK2879552 treatment resulted in a potent anti-proliferative growth effect in 19 of 25 AML cell lines (average EC50 = 38 nM), representing a range of AML subtypes. Potent growth inhibition was also observed on AML blast colony forming ability in 4 out of 5 bone marrow samples derived from primary AML patient samples (average EC50 = 205 nM). The effects of LSD1 inhibition were further characterized in an in vivo mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and upon engraftment, the mice were treated for 17 days. After 17 days of treatment, control treated mice had 80% GFP+ cells in the bone marrow whereas treated mice possessed 2.8% GFP positive cells (p<0.012). The percentage of GFP+ cells continued to decrease to 1.8% by 1-week post therapy. Remarkably, in a preliminary assessment for survival, control-treated mice succumbed to AML by 28 days post transplant, while treated mice showed prolonged survival. Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. GSK2879552 is currently in late preclinical development and clinical trials are anticipated to start in 2014. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Disclosures: Kruger: GlaxoSmithKline Pharmaceuticals: Employment. Mohammad:GlaxoSmithKline Pharmaceuticals: Employment. Smitheman:GlaxoSmithKline Pharmaceuticals: Employment. Liu:GlaxoSmithKline Pharmaceuticals: Employment. Pappalardi:GlaxoSmithKline Pharmaceuticals: Employment. Federowicz:GlaxoSmithKline Pharmaceuticals: Employment. Van Aller:GlaxoSmithKline Pharmaceuticals: Employment. Kasparec:GlaxoSmithKline Pharmaceuticals: Employment. Tian:GlaxoSmithKline Pharmaceuticals: Employment. Suarez:GlaxoSmithKline Pharmaceuticals: Employment. Rouse:GlaxoSmithKline Pharmaceuticals: Employment. Schneck:GlaxoSmithKline Pharmaceuticals: Employment. Carson:GlaxoSmithKline Pharmaceuticals: Employment. McDevitt:GlaxoSmithKline Pharmaceuticals: Employment. Ho:GlaxoSmithKline Pharmaceuticals: Employment. McHugh:GlaxoSmithKline Pharmaceuticals: Employment. Miller:GlaxoSmithKline Pharmaceuticals: Employment. Johnson:GlaxoSmithKline Pharmaceuticals: Employment. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other. Tummino:GlaxoSmithKline Pharmaceuticals: Employment.

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Repositioning of Quinacrine for Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.3609.3609 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3609-3609

Author(s):

Anna Eriksson ◽

Albin Osterros ◽

Sadia Hassan ◽

Joachim Gullbo ◽

Linda Rickardson ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Cytotoxic Activity ◽

Tumor Cells ◽

Cell Lines ◽

Myeloid Leukemia ◽

Lymphocytic Leukemia ◽

Ribosomal Biogenesis ◽

Rheumatic Drug ◽

Acute Myeloid

Abstract Background: A promising strategy for new drug discovery is ‘repositioning’, in which a new indication for an existing drug is identified. Using this approach, known on-patent, off-patent, discontinued and withdrawn drugs with unrecognized cancer activity, can be rapidly advanced into clinical trials for the new indication. We here report findings from a library screen of pharmacologically active and mechanistically annotated compounds in leukemia cells from patients aiming at the identification of repositioning candidates. Methods and results: The LOPAC®, 1280substance library (Sigma-Aldrich), with 1266 mechanistically annotated compounds, were investigated for cytotoxic activity by the fluorometric microculture cytotoxicity assay (FMCA) on tumor cells from 12 patients with leukemia (4 acute lymphocytic leukemia, 4 acute myeloid leukemia [AML], 4 chronic lymphocytic leukemia), as well as on peripheral blood mononuclear cells (PBMC) from 4 healthy donors. Sixty-eight compounds were identified as hits, defined as having a cytotoxic activity (less than 50% cell survival compared with controls) in all leukemia subgroups at the 10µM drug concentration used for screening. Only one of the hit compounds, quinacrine, showed higher activity in the leukemic cells than in normal PBMCs and was therefore selected for further preclinical evaluation focusing on AML. The aminoacridine quinacrine has a wide range of biological and therapeutical applications, and has been used for decades outside hemato-oncology, notably as an anti-protozoal and anti-rheumatic drug. Its side effects and toxicity are well characterized. Quinacrine showed significant cytotoxic activity in all four AML cell lines tested (HL-60, Kasumi-1, KG1a and MV4-11). In tumor cells from another 9 patients with AML, the cytotoxic effect (IC50 median 1.8, range 0.8-4 µM) was significantly superior to that in normal lymphocytes and clearly dose-dependent. Analysis of quinacrine data from the National Cancer Institute growth inhibitory screen in 60 cell lines (NCI 60 GI 50 data) was performed with the help of the NCI Cellminer database (http://discover.nci.nih.gov/cellminer/), and indicated leukemia sensitivity. To examine the ability of quinacrine to reverse diagnosis-specific gene expression, we utilized the Nextbio bioinformatics software, with its gene expression signatures of drug exposed myeloid leukemia cell cultures (HL60). These queries showed that myeloid leukemias had high reversibility scores. Moreover, gene enrichment and drug correlation data revealed a strong association to ribosomal biogenesis nucleoli. Translation initiation was observed including a high drug-drug correlation with ellipticine, a known inhibitor of RNA polymerase I (Pol-I). To validate the latter results, gene expression analysis of HL-60 cells exposed to quinacrine were obtained using the protocol described by Lamb et al (Science, 2006, 313, 1929), showing down regulation of Pol-1 associated RNA. Supporting these findings, quinacrine induced early inhibition of protein synthesis. Conclusions: The anti-protozoal and anti-rheumatic drug quinacrine has significant in vitro activity in AML. The anti-leukemic effect may be mediated by targeting ribosomal biogenesis. Considering its favorable and well-known safety profile, clinical studies of quinacrine in AML should be considered. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v124.21.915.915 ◽

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.

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A Rapid Culture Technique Produces Functional Dendritic-Like Cells from Human Acute Myeloid Leukemia Cell Lines

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/172965 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Jian Ning ◽

David Morgan ◽

Derwood Pamphilon

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Cell Surface ◽

Cell Lines ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Antigen Uptake ◽

Human Acute Myeloid Leukemia ◽

Ifn Γ ◽

Acute Myeloid

Most anti-cancer immunotherapeutic strategies involving dendritic cells (DC) as vaccines rely upon the adoptive transfer of DC loaded with exogenous tumour-peptides. This study utilized human acute myeloid leukemia (AML) cells as progenitors from which functional dendritic-like antigen presenting cells (DLC) were generated, that constitutively express tumour antigens for recognition by CD8+T cells. DLC were generated from AML cell lines KG-1 and MUTZ-3 using rapid culture techniques and appropriate cytokines. DLC were evaluated for their cell-surface phenotype, antigen uptake and ability to stimulate allogeneic responder cell proliferation, and production of IFN-γ; compared with DC derived from normal human PBMC donors. KG-1 and MUTZ-3 DLC increased expression of CD80, CD83, CD86, and HLA-DR, and MUTZ-3 DLC downregulated CD14 and expressed CD1a. Importantly, both KG-1 and MUTZ-3-derived DLC promoted proliferation of allogeneic responder cells more efficiently than unmodified cells; neither cells incorporated FITC-labeled dextran, but both stimulated IFN-γ production from responding allogeneic CD8+T cells. Control DC produced from PBMC using the FastDC culture also expressed high levels of critical cell surface ligands and demonstrated good APC function. This paper indicates that functional DLC can be cultured from the AML cell lines KG-1 and MUTZ-3, and FastDC culture generates functional KG-1 DLC.

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