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

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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The PARP Inhibitor Olaparib Antagonizes Leukemic Growth Induced By TET1 Overexpression in AML1-ETO Positive Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.4063.4063 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4063-4063 ◽

Cited By ~ 1

Author(s):

Shiva Bamezai ◽

Jing He ◽

Deniz Sahin ◽

Fabian Mohr ◽

Fabio Ciccarone ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Growth ◽

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Parp Inhibitor ◽

Leukemic Cell ◽

Aberrant Expression ◽

Acute Myeloid

Abstract DNA methylation patterns are highly deregulated in human acute myeloid leukemia (AML) cases and stratify AML patient samples into different subgroup. AML1-ETO is the most commonly occurring fusion gene in AML and these AML cases exhibit an aberrant and distinct methylation pattern. So far, the underlying mechanisms for this are only poorly understood. The TET1 dioxygenase has recently emerged as an important epigenetic modifier: by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) TET1 plays an important role in active demethylation, thereby regulating a variety of biological processes. It was linked to tumorigenesis based on the observation that its expression is frequently deregulated in solid cancer. However, the role of TET1 in AML1-ETO+ (AE+)human AML cases is yet unexplored. Using quantitative real time (qRT)- PCR we now show that AE+ AML is characterized by high and aberrant expression of TET1: the gene was significantly higher expressed in the majority of AE+ patients (n=7, p<0.01) compared to other AML subtypes such as inv(16) (n=11), PML-RARα+ (n=31), cytogenetically normal (CN)-AML patients (n=33) and CD34+ normal BM cells (n=4). This observation was consistent with published cDNA microarray data on large patient cohorts (Haferlach et al., JCO 2010, p<0.008 t-test, p<0.01 Anova) and recently published transcriptome data (TCGA) of AML patients. In contrast to TET1, TET2 and TET3 did not show significant higher expression in AE+ patients compared to other AML subtypes. In line with patient data, TET1 was highest expressed in the AE+ AML cell line KASUMI-1 and SKNO-1 compared to other AML cell lines (p<0.05 and n=3). Compared to normal CD34+ and myeloid (CD33+, CD15+ and CD14+) cells (n=3), TET1 was 10-fold and 16-fold higher expressed in AE+ patient samples (n= 7). Aberrant expression of TET1 in AE+ leukemic cells was associated with hypomethylation of its promoter and enrichment for H3K4me3 euchromatic marks at its promoter as determined by LC/MS and ChIP-qPCR respectively. Knockdown (KD) of TET1 mRNA using two short hairpin RNAs (shRNAs) in AE+ AML cell lines impaired their cell growth and clonogenicity by over 50% in vitro (n=3 and p<0.01). shRNA mediated depletion of TET1 did not impact the cell growth and clonogenicity of the TET1 negative cell line RAJI, ruling out off target effects of the shRNAs (n=3). In mice, KD of Tet1 in leukemic bone marrow cells expressing the truncated leukemogenic AML1-ETO9a (AE9a) fusion, dramatically inhibited cell growth (>60% compared to scrambled, n=3, p<0.01), clonogenicity (>50-70% reduction in primary CFCs, p<0.01, n=3) and importantly delayed onset of leukemia in vivo (median survival 35 days for scr vs 80 days for shRNA mice, n=4/arm, p<0.03). Tet1-knock-out c-kit+ hematopoietic stem and progenitor cells (HSPCs) transduced with AE9a showed reduced primary colony formation and impaired serial replanting capacity in vitro compared to AE9a transduced Tet1-wild-type HSPCs (>50% and >70%, respectively; p<0.001, n=3). Global analysis of 5hmC and 5mC levels using hMeDIP/MeDIP-seq performed on TET1 depleted KASUMI-1 cells revealed lower global 5hmC levels and increase in 5mC as compared to cells transduced with scrambled control (n=2). 3324 promoter regions lost 5hmC and gained 5mC upon TET1 depletion (-5kTSS, Fold enrichment cut off <2-fold, q-value<1e5). Recent studies have shown that PARP activity induces TET1 expression by regulating its promoter epigenetically. We could show that aberrant TET1 expression could be antagonized by the PARP inhibitor olaparib in AE+ leukemic cell lines. Furthermore, olaparib treatment decreased 5hmC levels and reduced cell growth and clonogenicity of human AE+ cell lines and of the murine AE9a+ leukemic cell line in vitro (n=3, p<0.01). In conclusion, our data indicates that aberrant TET1 expression contributes to the growth of AE+ AML by maintaining the 5-hydroxymethylome and that the PARP inhibitor olaparib can at least partially antagonize the oncogenic effect of TET in AML. Disclosures Mulaw: NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

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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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Distinct association of RUNX family expression with genetic alterations and clinical outcome in acute myeloid leukemia

Cancer Biomarkers ◽

10.3233/cbm-200016 ◽

2020 ◽

Vol 29 (3) ◽

pp. 387-397

Author(s):

Yangli Zhao ◽

Tingjuan Zhang ◽

Yangjing Zhao ◽

Jingdong Zhou

Keyword(s):

Acute Myeloid Leukemia ◽

Clinical Outcome ◽

Myeloid Leukemia ◽

Disease Free Survival ◽

Genetic Alterations ◽

Differentially Expressed ◽

Aberrant Expression ◽

Hematopoietic Stem ◽

Frequent Event ◽

Acute Myeloid

BACKGROUND: The runt-related transcription factor family (RUNXs) including RUNX1, RUNX2, and RUNX3 are key transcriptional regulators in normal hematopoiesis. RUNXs dysregulations caused by aberrant expression or mutation are frequently seen in various human cancers especially in acute myeloid leukemia (AML). OBJECTIVE: We systemically analyzed the expression of RUNXs and their relationship with clinic-pathological features and prognosis in AML patients. METHODS: Expression of RUNXs was analyzed between AML patients and normal controls from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) projects. Correlations between RUNXs expression and clinical features together with survival were further analyzed. RESULTS: All RUNXs expression in AML patients was significantly increased as compared with controls. RUNXs expression was found to be significantly associated with genetic abnormalities such as RUNX1 mutation, t(8;21) and inv(16)/t(16;16). By Kaplan-Meier analysis, only RUNX3 overexpression was associated with shorter overall survival (OS) and disease-free survival (DFS) among non-M3 AML patients. Notably, in high RUNX3 expression groups, patients received hematopoietic stem cell transplantation (HSCT) had markedly better OS and DFS than patients without HSCT among both all AML and non-M3 AML. In low RUNX3 expression groups, there were no significant differences in OS and DFS between HSCT and non-HSCT groups among both all AML and non-M3 AML. In addition, a total of 835 differentially expressed genes and 69 differentially expressed microRNAs were identified to be correlated with RUNX3 expression in AML. CONCLUSION: RUNXs overexpression was a frequent event in AML, and was closely associated with diverse genetic alterations. Moreover, RUNX3 expression may be associated with clinical outcome, and helpful for guiding treatment choice between HSCT and chemotherapy in AML.

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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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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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MYB Overexpression Is Directly Involved in Acute Myeloid Leukemia Pathogenesis and Could Constitute a New Therapeutic Target for Patients with Aberrant Expression of This Gene.

Blood ◽

10.1182/blood.v114.22.2402.2402 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2402-2402 ◽

Cited By ~ 1

Author(s):

Carmen Vicente ◽

Ana Conchillo ◽

Daphnie Pauwels ◽

Iria Vazquez ◽

Laura Garcia-Orti ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Complete Remission ◽

Synergistic Effect ◽

Cell Lines ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Recurrent Event ◽

Aberrant Expression ◽

Acute Myeloid

Abstract Abstract 2402 Poster Board II-379 The MYB proto-oncogene encodes a nuclear transcription factor with an essential role in proliferation, lineage commitment, and differentiation of hematopoietic progenitor cells. Proper levels of MYB are known to be important during hematopoietic cell development, and the Myb gene is a frequent target of retroviral insertions in myeloid, B- and T-cell leukemias in the mouse. Overexpression of MYB in T-acute lymphoblastic leukemia (T-ALL) causes a differentiation block of the T cells, and it has been shown that NOTCH1 mutation and MYB duplication cooperate in the pathogenesis of T-ALL. Our aim was to study the role of MYB in the pathogenesis of acute myeloid leukemia (AML), and to investigate its potential as a target for therapy. We functionally characterized MYB in 15 AML cell lines. Twelve of the 15 cell lines tested had MYB overexpression. Knockdown of MYB by siRNA in these cell lines caused decreased cell viability and proliferation, and reduced the clonogenic capacity, that could be explained in some cell lines by changes on the stage of cell differentiation. These results show that MYB overexpression is involved in the pathogenesis of AML. Moreover, knockdown of MYB in combination with common AML treatments (Idarubicin, Cytarabine and Sorafenib) had a strong synergistic effect on proliferation and viability of cells, suggesting that MYB could be a new target for therapy in AML. These observations prompted us to quantify MYB expression in a cohort of 159 patients with AML at diagnosis. We detected MYB overexpression in 14.5% (23/159) patients, with a higher prevalence within the intermediate prognosis group (17/83, 20.5%), particularly in patients with normal karyotype (NK) (14/62, 22.6%). Interestingly, 33% of patients without FLT-3 ITD and NPM1 mutations had MYB overexpression. To study the prognosis impact of MYB overexpression in AML, we performed a survival analysis in a preliminary series of 100 AML patients at diagnosis. As expected, significant differences in OS according to age, complete remission and cytogenetic prognostic group were found (p<0.01). MYB overexpression had no significant impact in the OS; however, this genetic marker allowed distinguishing a group of patients with a worse outcome within the group that did not get complete remission after treatment. Recently it has been described that MYB duplication causes elevated MYB expression in T-ALL; we detected duplication of MYB in 2 of 13 AML cell lines and in 2 patients with MYB overexpression (2/23, 8.6%). In conclusion, these results show that aberrant expression of MYB is involved in the activation of pathways responsible for the increased proliferative and clonogenic capacity that is characteristic of AML, independently of other genetic aberrations. Moreover, we show that MYB overexpression is a recurrent event in AML, especially in the subgroup of patients with NK, and that MYB could cooperate with other mutations in the leukemic transformation, as described previously in T-ALL. The synergistic effect of combined treatments with MYB knockdown, suggest that MYB silencing could be a new target for therapy in patients with AML and MYB overexpression. 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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In vitro activity of a G-quadruplex-stabilizing small molecule that synergizes with Navitoclax to induce cytotoxicity in acute myeloid leukemia cells

BMC Cancer ◽

10.1186/s12885-019-6464-9 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 7

Author(s):

Justin J. Montoya ◽

Megan A. Turnidge ◽

Daniel H. Wai ◽

Apurvi R. Patel ◽

David W. Lee ◽

...

Keyword(s):

Dna Damage ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Small Molecule ◽

Myeloid Leukemia ◽

In Vitro Activity ◽

Combination Drug ◽

G Quadruplex ◽

Acute Myeloid

Abstract Background Acute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that arise from genomic alterations in the expression of key growth regulatory genes causing cells to assume an undifferentiated state and continue to proliferate. Recent efforts have focused on developing therapies that target specific protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target and thought to be “undrugable” because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A novel technology that circumvents some of these issues is the use of small molecules that stabilize secondary DNA structures present in the promoters of many potential oncogenes and modulate their transcription. Methods This study characterizes the in vitro activity of the G-quadruplex-stabilizing small molecule GQC-05 in AML cells. The effect of GQC-05 on three AML cell lines was analyzed using viability and apoptosis assays. GQC-05 has been shown to down-regulate MYC through G-quadruplex stabilization in Burkitt’s lymphoma cell lines. MYC expression was evaluated through qPCR and immunoblotting in the three AML cell lines following the treatment of GQC-05. In order to identify other therapeutic agents that potentiate the activity of GQC-05, combination drug screening was performed. The drug combinations were validated using in vitro cytotoxicity assays and compared to other commonly used chemotherapeutic agents. Results GQC-05 treatment of KG-1a, CMK and TF-1 cells decreased cell viability and resulted in increased DNA damage and apoptosis. Additionally, treatment of KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with a more pronounced effect in KG-1a cells. Combination drug screening identified the Bcl-2/Bcl-XL inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed a synergistic decrease in cell viability of AML cells as determined by Chou-Talalay analysis, and induced more DNA damage, apoptosis, and rapid cytotoxicity. The cytotoxicity induced by GQC-05 and Navitoclax was more potent than that of Navitoclax combined with either cytarabine or doxorubicin. Conclusion These results suggest that the G-quadruplex stabilizing small molecule GQC-05 induces down regulated MYC expression and DNA damage in AML cells. Treatment with both GQC-05 with a Bcl-2/Bcl-XL inhibitor Navitoclax results in increased cytotoxic activity, which is more pronounced than Navitoclax or GQC-05 alone, and more significant than Navitoclax in combination with cytarabine and doxorubicin that are currently being used clinically.

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