Synergistic Reactivation Of Cancer/Testis Antigens By Combination Treatment With Decitabine and Histone Deacetylase Inhibitors (Valproate, Panobinostat) In Myeloid Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3763-3763
Author(s):  
Nadja Blagitko-Dorfs ◽  
Tobias Bauer ◽  
Maren Prinz ◽  
Wolfram Brugger ◽  
Gesine Bug ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Myeloid Leukemia ◽  
Histone Deacetylase Inhibitors ◽  
Leukemia Cells ◽  
Cancer Testis Antigens ◽  
Dose Dependent ◽  
Hdaci Treatment ◽  
Cancer Testis

Abstract Introduction Epigenetic therapies with azanucleoside DNA hypomethylating agents, alone or in combination with histone deacetylase inhibitors (HDACi), show clinical activity in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), particularly when given at non-cytotoxic doses. They are able to reactivate epigenetically silenced genes including, among others, a number of highly immunogenic proteins dubbed Cancer/testis antigens (CTAs), predominantly the CTAs located on the X chromosome. We have previously shown that decitabine can induce expression of several CTAs, including MAGEB2 and NY-ESO-1, in myeloid cells in vitro and thereby trigger an immune response (Almstedt et al., Leuk. Res. 2010). Induction of a CTA-specific cytotoxic T cell response in vivo was reported also in AML patients treated with azacitidine and sodium valproate (VPA) and correlated with clinical response (Goodyear et al., Blood 2010). To the best of our knowledge, no data have yet been reported on the effect of combination treatment with decitabine and panobinostat or sodium valproate (VPA) on CTA reactivation in myeloid leukemia. Aim We hypothesized that by combining decitabine with HDACi we could further enhance expression of CTAs in myeloid leukemia cells and thereby boost recognition of the malignant cells by the cytotoxic T lymphocytes. Methods The myeloid cell lines U937 and Kasumi-1 were treated with decitabine alone or in combination with the HDACi VPA or panobinostat applied at non-toxic concentrations (>80% cell viability). Expression of CTAs was analyzed by RT-qPCR and Western blot after 48 hours of HDACi treatment. DNA methylation of NY-ESO-1 and MAGEB2 promoter regions was quantified by pyrosequencing. Bone marrow mononuclear cells from 19 AML patients (treated with or without VPA as add-on to decitabine in the ongoing randomized phase II DECIDER clinical trial, NCT00867672) were collected before and on day 15 of treatment, in some patients also after 2 treatment cycles. CTA mRNA expression and promoter DNA methylation were quantified as described above. Results VPA or panobinostat alone did not induce MAGEB2 or NY-ESO-1 expression in vitro. However the pretreatment of cells with decitabine prior to addition of either HDACi resulted in a synergistic dose-dependent reactivation of MAGEB2 and NY-ESO-1 on the mRNA level (confirmed for the latter on the protein level). Pyrosequencing analysis of the heavily methylated NY-ESO-1 and MAGEB2 promoters revealed, as expected, no methylation changes upon HDACi treatment, but a dose-dependent hypomethylation upon decitabine. In recently initiated in vivo studies (DECIDER trial), until now cells from 19 AML patients receiving epigenetic treatment were sequentially analyzed. Induction of MAGEB2 mRNA was observed in 9 patients (from absent to a median of 0.002 relative to GAPDH, range 0.0004-0.043), with concomitant DNA hypomethylation of the MAGEB2 promoter from median 83% pretreatment methylation (range 63%-90%) to 63% posttreatment (range 44%-74%). In 5 patients modest hypomethylation without changes in MAGEB2 expression was observed (from median pretreatment values of 89% [72%-92%] to 82% [58%-87%] posttreatment). Another 5 patients disclosed neither hypomethylation nor reexpression of MAGEB2 (results as yet blinded to treatment arm and clinical response). Conclusions Combined epigenetic treatment with the hypomethylating agent decitabine and the HDACi VPA or panobinostat synergistically induced a dose-dependent reactivation of the CTAs MAGEB2 and NY-ESO-1 in vitro, accompanied by promoter hypomethylation. First translational results of the DECIDER AML trial also indicate in vivo effects of the epigenetic treatment on CTA induction. The unmasking of CTAs to the immune system by epigenetically active drugs can increase anti-tumor immune responses, and thus has clear implications for future clinical trials combining epigenetic therapy and specific immunotherapy in myeloid neoplasia. Disclosures: No relevant conflicts of interest to declare.

siRNA/lipopolymer nanoparticles to arrest growth of chronic myeloid leukemia cells in vitro and in vivo

2018 ◽  
Vol 130 ◽  
pp. 66-70 ◽  
Author(s):  
Juliana Valencia-Serna ◽  
Hamidreza M. Aliabadi ◽  
Adam Manfrin ◽  
Mahsa Mohseni ◽  
Xiaoyan Jiang ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Arrest Growth

scholarly journals HDAC3 Activity is Essential for Human Leukemic Cell Growth and the Expression of β-catenin, MYC, and WT1

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1436 ◽  
Author(s):  
Mandy Beyer ◽  
Annette Romanski ◽  
Al-Hassan M. Mustafa ◽  
Miriam Pons ◽  
Iris Büchler ◽  
...  
Keyword(s):  
Dna Damage ◽  
Myeloid Leukemia ◽  
Histone Deacetylase Inhibitors ◽  
Wilms Tumor ◽  
Leukemic Cell ◽  
Replication Stress ◽  
Leukemic Cells ◽  
Hdac6 Inhibitor

Therapy of acute myeloid leukemia (AML) is unsatisfactory. Histone deacetylase inhibitors (HDACi) are active against leukemic cells in vitro and in vivo. Clinical data suggest further testing of such epigenetic drugs and to identify mechanisms and markers for their efficacy. Primary and permanent AML cells were screened for viability, replication stress/DNA damage, and regrowth capacities after single exposures to the clinically used pan-HDACi panobinostat (LBH589), the class I HDACi entinostat/romidepsin (MS-275/FK228), the HDAC3 inhibitor RGFP966, the HDAC6 inhibitor marbostat-100, the non-steroidal anti-inflammatory drug (NSAID) indomethacin, and the replication stress inducer hydroxyurea (HU). Immunoblotting was used to test if HDACi modulate the leukemia-associated transcription factors β-catenin, Wilms tumor (WT1), and myelocytomatosis oncogene (MYC). RNAi was used to delineate how these factors interact. We show that LBH589, MS-275, FK228, RGFP966, and HU induce apoptosis, replication stress/DNA damage, and apoptotic fragmentation of β-catenin. Indomethacin destabilizes β-catenin and potentiates anti-proliferative effects of HDACi. HDACi attenuate WT1 and MYC caspase-dependently and -independently. Genetic experiments reveal a cross-regulation between MYC and WT1 and a regulation of β-catenin by WT1. In conclusion, reduced levels of β-catenin, MYC, and WT1 are molecular markers for the efficacy of HDACi. HDAC3 inhibition induces apoptosis and disrupts tumor-associated protein expression.

CREB Plays a Critical Role in the Regulation of Normal and Malignant Hematopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1224-1224
Author(s):  
Jerry C. Cheng ◽  
Dejah Judelson ◽  
Kentaro Kinjo ◽  
Jenny Chang ◽  
Elliot Landaw ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Leukemia Cells ◽  
Mouse Bone Marrow ◽  
T315i Mutation

Abstract The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p<0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p<0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.

Rig-I Activates Expression of Interferon-Stimulated Genes (ISGs) and Inhibits the Proliferation of Acute Myeloid Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2846-2846 ◽  
Author(s):  
Nan-Nan Zhang ◽  
Lei Chen ◽  
Wu Zhang ◽  
Xian-Yang Li ◽  
Lin-Jia Jiang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Terminal Part ◽  
Granulocytic Differentiation ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Acute promyelocytic leukemia (APL) is initiated by the formation of PML/RARα oncogenic fusion protein, a potent transcriptional repressor. Retinoid acid (RA) at pharmacological dosage can physically bind to the PML/RARα protein, ushering in the unfolding of downstream programs normally regulated by the wild type RARα. However, through what particular regulatory pathways RA inhibits APL malignant hematopoiesis has remained largely obscured. Rig-I is one of the genes whose mRNA levels were highly up-regulated, along with all-trans-RA (ATRA)-induced terminal granulocytic differentiation of APL cell line NB4 cells in vitro. Based on the analysis of a Rig-I−/− mouse model, recently we have reported a critical regulatory role of Rig-I in normal granulopoiesis. To understand the functional contribution of Rig-I induction in RA-mediated leukemia cell differentiation, we converted a pair of previously reported Rig-I RNAi-duplex sequences into a miR30a-based small hairpin-encoding sequence, which was expressed under the CMV enhancer/promoter within a lentiviral vector. As expected, Rig-I shRNAmir30 infection induced a significant knockdown of Rig-I protein level, and accordingly its delivery into HL-60 cells partially inhibited ATRA-induced granulocytic differentiation, growth inhibition/cell cycle arrest and apoptosis induction, suggesting that Rig-I upregulation participates in RA-induced granulocytic differentiation of acute myeloid leukemia cells. In order to investigate the effect of Rig-I induction on the proliferation of APL cells in vivo, we transduced PML/RARα-harboring leukemic cells with vector or Rig-I-expressing retrovirus, and then transplanted these cells into the syngeneic mice. The vector-transduced APL cells readily expanded in vivo, but the proliferation of Rig-I-transduced cells was apparently prohibited. Moreover, we found that the forced expression of Rig-I induced the expression of numerous ISGs in APL cells, which was recapitulated by the transduction of the C terminal part of Rig-I, but not by the N terminal part. In line with this, during the in vitro short-term culture post-IFNγ or IFNα stimulation, Stat1 phosphorylation at p701 in Rig-I−/− granulocytes was significantly inhibited. In parallel, the induction of multiple ISGs by IFNs was also significantly impaired. In conclusion, our findings indicate that the Rig-I induction inhibited APL reconstitution potentially through up-regulating a number of ISGs via regulating Stat1Tyr701 phosphorylation.

GFI1 Is a Tumor Suppressor in Myeloid Progenitors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2942-2942
Author(s):  
Aditya Chaubey ◽  
Shane Hormon ◽  
Chinavenmeni S. Velu ◽  
Tristan Bourdeau ◽  
Jinfang Zhu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Myeloid Leukemia ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Increased Risk ◽  
Myeloid Leukemias ◽  
Dose Dependent ◽  
Anterior Posterior

Abstract In severe congenital neutropenia (SCN) patients and mice with Growth factor independent-1 (Gfi1) loss of function, arrested progenitors are suspended in a hyperproliferative state while terminal granulpoiesis is blocked. SCN patients are at increased risk for the development of acute myeloid leukemia. We demonstrate that Gfi1 directly targets HoxA9, Pbx1 and Meis1 during normal myelopoiesis. Gfi1−/− progenitors exhibit elevated levels of HoxA9, Pbx1 and Meis1, exaggerated HoxA9-Pbx1-Meis1 activity, and increased persistence in vivo and in vitro. Limiting HoxA9 alleles corrects, in a dose dependent manner, in vivo and in vitro phenotypes observed with loss of Gfi1. Moreover, in a manner conserved in Drosophila anterior/posterior patterning, we demonstrate that these factors can compete for occupancy of DNA sequences encoding composite Gfi1-HoxA9-Pbx1-Meis1 binding sites. Finally, the expression of Gfi1 and HoxA9 are inverse and stratify human myeloid leukemias, suggesting a role for HoxA9- Gfi1 antagonism in human AML. In agreement with this, a myeloproliferative disorder progresses into a rapid, lethal and transplantable myeloid leukemia in a Gfi1−/− setting. We conclude that the lifespan and oncogenic transformation of hematopoietic progenitor cells is regulated through a conserved competition between Gfi1 and HoxA9-Pbx1-Meis1.

The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3090-3090 ◽  
Author(s):  
Folashade Otegbeye ◽  
Nathan Mackowski ◽  
Evelyn Ojo ◽  
Marcos De Lima ◽  
David N. Wald
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Ex Vivo ◽  
Leukemia Cells ◽  
Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

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