The Dose-Dependent Effects of Microrna-155 in Acute Myeloid Leukemia

Abstract MicroRNAs are a class of non-coding, regulatory RNAs that control several critical cellular processes. Subsets of microRNAs are dysregulated in cancer, and can act as oncogenes or tumour suppressors. MicroRNA-155 (miR-155) has a well-established role as an oncogene in B cell lymphoma but has a more enigmatic role in acute myeloid leukemia (AML), in which there is evidence that miR-155 may promote or repress the development and progression of AML. We have used enforced expression of miR-155 in murine AML cell lines and AML models to explore the biology of miR-155 in AML. We show that the capacity of miR-155 to promote or repress the ability of AML cells to form colonies and to proliferate is dependent on miR-155 expression levels. Enforced high expression of miR-155 in AML cell lines results in reduced proliferation and colony formation. However, critical long-term assays of cells transduced with miR-155 resulted in selection in favour of an intermediate miR-155 expression level accompanied by a restoration in clonogenic potential. In vivo, enforced expression of miR-155 in murine AML models showed no differences in disease latency compared to controls, but resulted in an increased tumour burden. Most interestingly, RNA-Sequencing analysis demonstrated that the contrasting levels of miR-155 regulate a substantially different set of gene targets, with downstream consequences on transcription that are consistent with the contrasting effects of high and intermediate miR-155 levels. The intermediate levels of miR-155 we observe are the same as that seen in human AML, whereas the high levels of miR-155 have a completely different inflammatory counterpart. Our data shows that that the levels of miR-155 powerfully influences that gene targets it controls and the resultant phenotypes observed. MiR-155 expressed within a specific range promotes AML disease progression. Disclosures No relevant conflicts of interest to declare.

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Epigenetic Inactivation of DBC1 in Acute Myeloid Leukaemia.

Blood ◽

10.1182/blood.v114.22.4429.4429 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4429-4429

Author(s):

Chen Zhao ◽

Aili Dai ◽

Ling Chen ◽

Xiaoping Sun ◽

Xin Han ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Mrna Expression ◽

Cell Lines ◽

Myeloid Leukemia ◽

Hematological Malignancies ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Lymphoblastic Leukemia ◽

B Cell Lymphoma ◽

Acute Myeloid

Abstract Abstract 4429 DNA hypermethylation has important implications in the tumorigenesis and prognosis in acute myeloid leukemia (AML). To identify relevant methylated genes in AML, we have compared several expression and methylation profilings. With expression analysis, we identified that TRPC6, DBC1, DCC and SOX9 have decreased expression levels in the most analyzed AML cell lines. Among these candidates, DBC1 (deleted bladder cancer 1), a putative tumor suppressor, drew our attention because it is frequently methylated not only in hematological malignancies, including diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and acute lymphoblastic leukemia, but also in epithelial cancers. DBC1 may play an important role in the regulation of cell growth and programmed cell death. But the mechanisms of transcriptional control and function role in the hematological malignancies, especially on acute myeloid leukemia, are not well known. In this study, we analyzed the DBC1 expression pattern in 9 AML cell lines with RT-PCR analysis. DBC1 mRNA expression was observed in normal bone-marrow but diminished expression in all of 9 AML cell lines. DBC1 methylation was frequently observed in AML cells (9 of 9, 100%) and inversely correlated with DBC1 mRNA expression in a COBRA analysis (Combined Bisulfite Restriction Analysis). We also detected a frequent methylation of DBC1 in primary AML patient samples (9 of 9, 100%). These findings indicate that DBC1 is frequently silenced by hypermethylation in AML. We are in the process of investigation the functional role of DBC1 in the pathogenesis. In addition, diagnostic and prognostic values of DBC1 in AML are being pursued.* Chen Zhao and Aili Dai contributed equally to the presented work. 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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Vgamma9Vdelta2 T Cells-Based Immunotherapy Targeting BTN3 Molecules in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.3726.3726 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3726-3726

Author(s):

Daniel Olive ◽

Audrey Benyamine ◽

Aude Le Roy ◽

Rémy Castellano ◽

Julie Gertner-Dardenne ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Mevalonate Pathway ◽

Conflicts Of Interest ◽

Tumor Burden ◽

Acute Myeloid

Abstract As they can kill Acute Myeloid Leukemia (AML) blasts in vitro and in vivo, Vg9Vd2T cells are key players in the design of new strategies of immunotherapy. AminoBisphonates (NBP) can enhance their activation in vitro and in vivo. Their combination with low-dose IL2 has shown promising results in 2 patients with AML who underwent partial remission. NBP treatment of blasts inhibits the Mevalonate pathway. The subsequent accumulation of Isopentenyl Diphosphate sensitize AML blasts to Vg9Vd2T cells killing but some AML cell lines blasts are resistant to this TCR mediated-lysis. Butyrophilin 3 A1 (BTN3A1) has been shown to be involved in IPP recognition and Vg9Vd2 T cells activation. Agonist monoclonal antibodies (mAb) recognizing the 3 isoforms of BTN3, can trigger BTN3 on tumor cell lines and sensitize them to Vg9Vd2 T cells lysis. We show that primary AML blasts from patient at diagnosis are heterogeneously killed by allogenic-IL-2-NBP-expanded Vg9Vd2 T. Some are resistant to this lysis and/or poorly sensitized by NBP. BTN3 molecules are highly expressed by blasts of AML cell lines and primary AML samples. We show that treatment of primary AML blasts with agonist anti-BTN3 mAb can overcome the resistance to Vg9Vd2 cells lysis in vitro. We assess this effect in vivo, showing that the addition of agonist anti-BTN3 mAb to Vg9Vd2 cells infusion decreased the tumor burden and increased the survival of NOG mice xenografted with luciferase-transduced U937 cell line. We confirm this effect in a model of mice xenografted with primary AML blasts, showing that treatment with anti-BTN3 mAb added to Vg9Vd2 cells infusion can decrease the number of blastic cells in the spleen, bone marrow and the blood, without requiring additional cytokine infusion. This drastic effect on sensitization of primary AML blasts to Vg9Vd2T cells killing could be of great interest especially in cases of refractory or relapsing AML. Disclosures No relevant conflicts of interest to declare.

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Extracellular ATP and CD39 Regulates Mitochondrial Function and Cytarabine Resistance through Intrinsic PKA-ATF-PGC1a Pathway in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-112534 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2737-2737

Author(s):

Jean-Emmanuel Sarry ◽

Christian Recher ◽

Nesrine Aroua

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Nucleoside Triphosphate ◽

Preclinical Model ◽

Acute Myeloid

Abstract Relapses in acute myeloid leukemia (AML) are caused by chemoresistant leukemic populations and new therapeutic approaches that specifically target these cells are urgently needed. Based on transcriptomic analyses of relevant PDX preclinical model of the resistance to cytarabine (AraC) and of the residual disease in patients, we identified ecto-nucleoside triphosphate diphosphohydrolase-1 CD39 (ENTPD1) overexpressed in the residual leukemic cells in vivo after chemotherapy. By flow cytometry, we confirmed that AraC increased cell surface CD39 expression in AML cell lines in vitro and in vivo as well as in 24 diverse patient-derived xenograft models. We further observed this increase in 100 patients at 35-days post-intensive chemotherapy compared to their respective diagnosis. Interestingly, high CD39 expression on AML patients was associated with a worse response to AraC in vivo. Furthermore, we showed that FACS-sorted CD39high AML cells had increased mitochondrial mass and activity, and were resistant to AraC in vitro and in vivo. We demonstrated that CD39 downstream signaling pathway was dependent on cAMP-PKA-PGC1a-TFAM axis and its inhibition by H89 sensitized AML cells to AraC through the inhibition of mitochondrial OxPHOS biogenesis and function. Finally, pharmacological inhibition of CD39 ATP hydrolase activity or genetic invalidation of CD39 protein using two inhibitors or shRNA markedly enhanced AraC cytotoxicity in AML cell lines and primary patient samples in vitro and in vivo. Together, these results indicate CD39 as a new player of the intrinsic chemoresistance pathway and a new therapeutic target to specifically overcome AraC resistance and eradicate these leukemic cells responsible for relapses in AML. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v118.21.581.581 ◽

2011 ◽

Vol 118 (21) ◽

pp. 581-581

Author(s):

Patrick Griffin ◽

Steffan T Nawrocki ◽

Takashi Satou ◽

Claudia M Espitia ◽

Kevin R. Kelly ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Mouse Model ◽

Cell Lines ◽

Myeloid Leukemia ◽

Kinase Inhibitor ◽

Standard Of Care ◽

Clonogenic Survival ◽

Preclinical Studies ◽

Acute Myeloid

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

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DNMT3A mutations Predict for Inferior Outcome in NPM1-Wildtype and Molecular Unfavorable Cytogenetically-Normal Acute Myeloid Leukemia: A Study of the German-Austrian AMLSG

Blood ◽

10.1182/blood.v118.21.415.415 ◽

2011 ◽

Vol 118 (21) ◽

pp. 415-415 ◽

Cited By ~ 1

Author(s):

Verena I. Gaidzik ◽

Richard F. Schlenk ◽

Peter Paschka ◽

Anja Stölzle ◽

Andrea Corbacioglu ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Negative Impact ◽

Epigenetic Modification ◽

Methylation Status ◽

Prognostic Impact ◽

Sequencing Analysis ◽

Mutational Status ◽

Acute Myeloid

Abstract Abstract 415 Background: Alteration of DNA methylation, a hallmark of epigenetic modification, is currently discussed as one important pathomechanism in leukemogenesis. Using a next-generation sequencing approach, a frameshift mutation of the gene encoding the DNA methyltransferase (DNMT3A) in an acute myeloid leukemia (AML) case was identified. DNMT3A catalyses the addition of a methyl group to the cytosine residue of CpG dinucleotides, thereby affecting promoter methylation status and gene expression. Subsequent sequencing analysis in an independent cohort of 288 AML patients (pts) revealed DNMT3A mutations (DNMT3Amut) in 22% of the pts; mutations were associated with intermediate-risk cytogenetics and poor outcome. Aims: To evaluate frequency and clinical impact of DNMT3Amut in pts with AML aged 18 to 61 years who were treated within AMLSG treatment trials AML HD98A (Schlenk et al., J Clin Oncol 2010;28:4642–8) and AMLSG 07–04 (NCT00151242). Methods: DNMT3A mutation analysis was performed in 1218 AML (HD98A, n=685; AMLSG 07–04, n=533; de novo AML, n=1102; s-AML, n=45; t-AML, n=69) using a DNA-based PCR assay for all coding exons (1 to 23) followed by direct sequencing. The median follow-up was 5.06 years. Results: DNMT3A mut were found with an overall frequency of 19.6% (239/1218); 189 mutations were located in the MTase domain clustering at amino acid R882 (79%). All but one mutation were heterozygous; only 4 cases had two mutations. DNMT3A sequence alterations included 17 frameshift, 4 nonsense, and 222 missense mutations. DNMT3A mut pts were significantly older (P=.01), more frequently females (P=.001), had higher white blood cell and platelet counts (both P<.0001), and higher bone marrow blasts percentage (P=.001). DNMT3Amut were associated with cytogenetically-normal AML (CN-AML, P<.0001), while DNMT3Amut were rare in favorable and adverse-risk karyotypes (P<.0001). Correlations with other molecular markers (NPM1, CEBPA, FLT3, IDH1/2, TET2, ASXL1) revealed a significant association with NPM1 (P<.0001), FLT3-ITD (P<.0001), and IDH1/2 (IDH1R132, P<.0001; IDH2R140, P=.0003; IDH2R172, P=.03) mutations, while co-occurrence of CEBPA (P=.02) and ASXL1 (P=.02) mutations was less frequent. DNMT3A mutational status did not impact complete remission (CR) rate, event-free (EFS) and relapse-free survival (RFS), neither in the whole cohort (P=.09, P=.98, P=.11; respectively) nor in the subgroup of CN-AML (P=.39, P=.79, P=.19, respectively). DNMT3Amut had a negative impact on overall survival (OS) in trend in the whole cohort (P=.07) and significantly in CN-AML (P=.02). In multivariable analyses, DNMT3Amut were in trend associated with a negative prognostic impact on OS (hazard ratio, 1.24; P=.06). In addition, we performed subgroup analyses according to (1) the NPM1 mutational status, and (2) the molecular risk groups of CN-AML (as defined by the European LeukemiaNet classification). DNMT3Amut did not impact OS in NPM1-mutated patients in the whole cohort as well as in CN-AML (P=.34; P=.22; respectively), while in NPM1-wildtype patients DNMT3Amut were associated with inferior OS in both, the whole cohort and in CN-AML (P=.001; P=.005; respectively). In molecular unfavorable CN-AML (NPM1-wildtype with or without FLT3-ITD, NPM1-mutated with FLT3-ITD, CEBPA-wildtype), DNMT3Amut were significantly associated with worse OS (P=.002) compared with DNMT3A-wildtype pts, even outweighing FLT3-ITD as an unfavorable prognostic marker. There was no effect of DNMT3Amut in molecular favorable-risk CN-AML. Conclusions: DNMT3A mutations are confirmed as frequent genetic aberrations in AML, associated with normal karyotype, NPM1, FLT3-ITD, and IDH1/2 mutations. DNMT3Amut predicts for inferior outcome in molecularly-defined subsets of AML, that is, NPM1-wildtype AML and molecular unfavorable CN-AML. As a single marker, DNMT3Amut only had a moderate effect on outcome. Disclosures: No relevant conflicts of interest to declare.

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Activity of the Hypoxia-Activated Prodrug, TH-302, in Human Acute Myeloid Leukemia Models

Blood ◽

10.1182/blood.v120.21.3611.3611 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3611-3611

Author(s):

Scott Portwood ◽

Deepika Lal ◽

Yung-Chun Hsu ◽

Rodrigo Vargas ◽

Meir Wetzler ◽

...

Keyword(s):

Overall Survival ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Chemotherapeutic Agents ◽

Scid Mice ◽

Hypoxic Conditions ◽

Acute Myeloid

Abstract Abstract 3611 Recent evidence has demonstrated the bone marrow (BM) microenvironment, the principal site of acute myeloid leukemia (AML) initiation and expansion, is characterized by intrinsically low oxygen tension. Theoretically, such microenvironmental changes may lead to the selective outgrowth of AML clones which are “better adapted” to survive within a severely hypoxic microenvironment and/or may confer resistance to chemotherapeutic agents, similar to solid tumor cells. We report here that human AML cells (HL60, ML-2) cultured under chronic hypoxic conditions mimicking the marrow microenvironment (1% O2, 72 hours) exhibited reduced sensitivity to cytarabine-induced apoptosis as compared with normoxic cells, as determined by flow cytometric analysis, western blot analysis, and cell viability assays. Similar results were noted in primary AML samples treated with cytarabine under normoxic and hypoxic conditions in colony formation assays (n=3 samples, p=0.01). In order to improve upon chemotherapy outcomes, we investigated the effects of TH-302, a hypoxia-activated bromo-isophosphoramidate mustard prodrug, which is currently undergoing clinical trial evaluation in multiple tumor types. Treatment of AML cell lines (HL60, HEL) and primary AML samples with TH-302 (at doses ranging from 0.1– 5 mM, p values ranging from <0.05–0.0001) resulted in dose- and hypoxic-dependent inhibition of AML proliferation and apoptosis. In vivo TH-302 treatment significantly decreased disease burden, as measured by total animal bioluminescence, and prolonged overall survival in two systemic human AML xenograft models (HEL-luciferase, HL60-luciferase) (Figure 1). Immunohistochemical studies demonstrated that TH-302 treatment reduced numbers of hypoxic (pimonidazole-positive) cells within the leukemic marrow microenvironment. Because prior data in animal models has shown that AML progression within the marrow is associated with expansion of hypoxic BM areas, we examined the effects of TH-302 treatment on systemic AML growth when initiated early (prior to AML inoculation) or late (several days following AML engraftment) in the disease process. TH-302 was equally effective at both time points. Although anti-vascular therapy has been shown to enhance tumor hypoxia in other cancer types, we noted no synergistic or additive in vivo effects when TH-302 therapy was combined with sorafenib, an inhibitor of vascular endothelial growth factor receptors (VEGFR), in our models. TH-302 therapy administered for two weeks in non-leukemic and leukemia-engrafted mice was not associated with hematologic toxicities. In summary, our results demonstrate the anti-leukemic activity of TH-302 in preclinical AML models and suggest that the efficacy of this and other drugs for AML therapy may be uniquely affected by the BM microenvironment. Further clinical development of TH-302 and other hypoxia-targeted drugs for AML therapy are warranted. Based on our data, higher TH-302 doses and/or chronic drug administration may be needed for optimal in vivo anti-leukemic activity. Figure 1. Effects of TH-302 treatment on systemic AML growth and overall survival in HL60-luciferase engrafted SCID mice. Figure 1. Effects of TH-302 treatment on systemic AML growth and overall survival in HL60-luciferase engrafted SCID mice. Disclosures: No relevant conflicts of interest to declare.

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