Enzasataurin Enhances ATRA-induced Differentiation of Acute Myeloid Leukemia Cells

Abstract Background All-trans retinoic acid (ATRA) is considered to be the sole clinically useful differentiating agent in the treatment of acute myeloid leukemia (AML). However, it has been effective only in acute promyelocytic leukemia (APL) but not other subtypes of AML. Therefore, finding strategies to sensitize cells to ATRA may develop ATRA-based therapy in the treatment of non-APL AML patients. Methods Cell proliferation was assessed by cell growth. Cell death was evaluated by cell viability and Annexin-V assay. Cell differentiation was analyzed by CD11b expression and morphology. To explore the underlying mechanisms, we studied the role of PKCβ, MEK, ERK, AKT, PU.1, C/EBPβ and C/EBPε by Western-blotting analysis. Results In this study, a clinically achievable concentration of enzastaurin enhanced ATRA-induced differentiation of AML cell lines, HL-60 and U937 as well as non-APL AML primary cells, while it also restored ATRA sensitivity in ATRA-resistant cell line, HL-60Res. Mechanistically, in all these cell lines, enzastaurin-ATRA (enz-ATRA) enhanced the protein levels of PU.1, CCAAT/enhancer binding protein β (C/EBPβ) and C/EBPε. The activity of protein kinase C β (PKCβ) was suppressed by enz-ATRA treatment in HL-60 and HL-60Res cells. However, another PKCβ-selective inhibitor mimicked the cellular and molecular effects of enzastaurin only in HL-60 cells. Only in U937 cells, enz-ATRA activated MEK and ERK, and a MEK specific inhibitor suppressed enz-ATRA-triggered differentiation and reduced the protein levels of PU.1, C/EBPβ and C/EBPε. Enz-ATRA activated Akt in HL-60 and HL-60Res cells. However, an Akt inhibitor blocked enz-ATRA-triggered differentiation and restored the protein levels of PU.1, C/EBPβ and C/EBPε only in HL-60Res cells. Therefore, PKCβ inhibition, MEK/ERK and Akt activation are involved in enz-ATRA-induced differentiation in HL-60, U937 and HL-60Res cells, respectively by modulation of the protein levels of C/EBPβ, C/EBPε and PU.1. Conclusions Enzastaurin, at the clinically achievable concentration, enhances ATRA-induced differentiation of AML cells by PKCβ inhibition, MEK/ERK and Akt activation. This study may provide a potential therapeutic strategy for AML patients.

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Epigenetic silencing of UBXN8 contributes to leukemogenesis in t(8;21) acute myeloid leukemia

Experimental & Molecular Medicine ◽

10.1038/s12276-021-00695-8 ◽

2021 ◽

Author(s):

Erna Yang ◽

Wei Guan ◽

Desheng Gong ◽

Jieying Li ◽

Caixia Han ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Fusion Protein ◽

Cell Lines ◽

Promoter Region ◽

Myeloid Leukemia ◽

Specific Inhibitor ◽

Epigenetic Silencing ◽

Cycle Arrest ◽

Acute Myeloid

AbstractThe formation of the RUNX1-RUNX1T1 fusion protein, resulting from the t(8;21) translocation, is considered to be one of the initiating events of t(8;21) acute myeloid leukemia (AML). However, the mechanisms of the oncogenic mechanism of RUNX1-RUNX1T1 remain unclear. In this study, we found that RUNX1-RUNX1T1 triggers the heterochromatic silencing of UBXN8 by recognizing the RUNX1-binding sites and recruiting chromatin-remodeling enzymes to the UBXN8 promoter region. Decitabine, a specific inhibitor of DNA methylation, upregulated the expression of UBXN8 in RUNX1-RUNX1T1+ AML cell lines. Overexpression of UBXN8 inhibited the proliferation and colony-forming ability of and promoted cell cycle arrest in t(8;21) AML cell lines. Enhancing UBXN8 levels can significantly inhibit tumor proliferation and promote the differentiation of RUNX1-RUNX1T1+ cells in vivo. In conclusion, our results indicated that epigenetic silencing of UBXN8 via methylation of its promoter region mediated by the RUNX1-RUNX1T1 fusion protein contributes to the leukemogenesis of t(8;21) AML and that UBXN8 targeting may be a potential therapeutic strategy for t(8;21) AML.

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CREB Controls C/Ebpd to Impair Myeloid Differentiation Process in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.4801.4801 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4801-4801

Author(s):

Claudia Tregnago ◽

Elena Manara ◽

Valeria Bisio ◽

Chiara Borga ◽

Silvia Bresolin ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Line ◽

Myeloid Leukemia ◽

Control Cell ◽

Myeloid Differentiation ◽

Differentiation Process ◽

Cd11b Expression ◽

Protein Levels ◽

Acute Myeloid

Abstract Purpose. The cAMP response element binding protein (CREB) is a transcription factor documented to be crucial for normal and neoplastic hematopoiesis. Its overexpression has already been demonstrated to impair myelopoiesis and to aberrantly control cell proliferation, apoptosis and cell cycle progression, both in vitro and in vivo. Its protein overexpression has been found in patients with acute myeloid leukemia (AML) contributing to decrease survival. CREB guides the expression of more than 5000 different targets in a tissue/cell-type specific fashion, and to date its main network towards leukemogenesis remains unknown. Here we aim to identify through which genes CREB triggers AML by using a CREB overexpressing transgenic zebrafish which developed AML in adulthood. Patients and methods. We performed gene expression profile of RNA from the kidney marrow of 14-months old CREB-zebrafish (n=5) and control zebrafish (n=5). Principal component analysis was performed using Partek Genomic Suite software to integrate zebrafish AML signature with human signature found in pediatric AML to find common CREB target genes. AML cell lines were used to validate CREB targets in vitro. Results. By GEP analysis and integration data we found 20 differentially expressed genes in both the zebrafish and human leukemia. Several of them were involved in the myeloid differentiation process, such as JUN, FOS and C/EBPδ. We confirmed that C/EBPδ protein levels highy correlated with phosphoCREB levels in zebrafish tumor as well as in a cohort of 66 pediatric patients with AML (r=0.79). We silenced and overexpressed CREB in AML or healthy bone marrow primary cultures respectively, and found that C/EBPδ was controlled by CREB transcriptional activity. We took advantages of a previously created HL60(CREB-) cell line, where CREB protein levels was absent compared to the control cell line called HL60(CREB+), where CREB was overexpressed. HL60(CREB-) cell line expressed lowered C/EBPδ levels compared to HL60(CREB+). To investigate CREB's role on differentiation program, cell lines were treated with all-trans-retinoic acid (ATRA). The evaluation of CD11b expression revealed that HL60(CREB-) differentiated to a higher extent compared to HL60(CREB+).Enhanced myeloid differentiation was also confirmed by cell morphology examination. Then we silenced C/EBPδ gene in HL60(CREB+) cell line and treated with ATRA, confirming that C/EBPδ lowered expression increased CD11b expression, phenocopying the HL60(CREB-) behavior. Conclusion. These results indicated that C/EBPδ is a CREB target crucial for myeloid differentiation. The new role discovered for CREB proto-oncogene in blocking myeloid differentiation process opens for further opportunities with differentiation agents to cure AML. Disclosures No relevant conflicts of interest to declare.

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CD157 signaling promotes survival of acute myeloid leukemia cells and modulates sensitivity to cytarabine through regulation of anti-apoptotic Mcl-1

Scientific Reports ◽

10.1038/s41598-021-00733-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yuliya Yakymiv ◽

Stefania Augeri ◽

Cristiano Bracci ◽

Sara Marchisio ◽

Semra Aydin ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Ex Vivo ◽

Specific Inhibitor ◽

Predictive Marker ◽

Adp Ribosyl Cyclase ◽

Acute Myeloid Leukemia Cells ◽

The Impact ◽

Acute Myeloid

AbstractCD157/BST-1 (a member of the ADP-ribosyl cyclase family) is expressed at variable levels in 97% of patients with acute myeloid leukemia (AML), and is currently under investigation as a target for antibody-based immunotherapy. We used peripheral blood and bone marrow samples from patients with AML to analyse the impact of CD157-directed antibodies in AML survival and in response to cytarabine (AraC) ex vivo. The study was extended to the U937, THP1 and OCI-AML3 AML cell lines of which we engineered CD157-low versions by shRNA knockdown. CD157-targeting antibodies enhanced survival, decreased apoptosis and reduced AraC toxicity in AML blasts and cell lines. CD157 signaling activated the PI3K/AKT/mTOR and MAPK/ERK pathways and increased expression of Mcl-1 and Bcl-XL anti-apoptotic proteins, while decreasing expression of Bax pro-apoptotic protein, thus preventing Caspase-3 activation. The primary CD157-mediated anti-apoptotic mechanism was Bak sequestration by Mcl-1. Indeed, the Mcl-1-specific inhibitor S63845 restored apoptosis by disrupting the interaction of Mcl-1 with Bim and Bak and significantly increased AraC toxicity in CD157-high but not in CD157-low AML cells. This study provides a new role for CD157 in AML cell survival, and indicates a potential role of CD157 as a predictive marker of response to therapies exploiting Mcl-1 pharmacological inhibition.

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Antitumor Effect of Pomolic Acid in Acute Myeloid Leukemia Cells Involves Cell Death, Decreased Cell Growth and Topoisomerases Inhibition

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180412120128 ◽

2019 ◽

Vol 18 (10) ◽

pp. 1457-1468

Author(s):

Michelle X.G. Pereira ◽

Amanda S.O. Hammes ◽

Flavia C. Vasconcelos ◽

Aline R. Pozzo ◽

Thaís H. Pereira ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Natural Compound ◽

Dna Topoisomerases ◽

Human Dna ◽

Pomolic Acid ◽

Acute Myeloid

Background: Acute myeloid leukemia (AML) represents the largest number of annual deaths from hematologic malignancy. In the United States, it was estimated that 21.380 individuals would be diagnosed with AML and 49.5% of patients would die in 2017. Therefore, the search for novel compounds capable of increasing the overall survival rate to the treatment of AML cells is urgent. Objectives: To investigate the cytotoxicity effect of the natural compound pomolic acid (PA) and to explore the mechanism of action of PA in AML cell lines with different phenotypes. Methods: Three different AML cell lines, HL60, U937 and Kasumi-1 cells with different mechanisms of resistance were used to analyze the effect of PA on the cell cycle progression, on DNA intercalation and on human DNA topoisomerases (hTopo I and IIα) in vitro studies. Theoretical experiments of the inhibition of hTopo I and IIα were done to explore the binding modes of PA. Results: PA reduced cell viability, induced cell death, increased sub-G0/G1 accumulation and activated caspases pathway in all cell lines, altered the cell cycle distribution and inhibited the catalytic activity of both human DNA topoisomerases. Conclusion: Finally, this study showed that PA has powerful antitumor activity against AML cells, suggesting that this natural compound might be a potent antineoplastic agent to improve the treatment scheme of this neoplasm.

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Chemotherapy-Induced Survivin Regulation in Acute Myeloid Leukemia Cells

Applied Sciences ◽

10.3390/app11010460 ◽

2021 ◽

Vol 11 (1) ◽

pp. 460

Author(s):

Petra Otevřelová ◽

Barbora Brodská

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Survivin Expression ◽

Mitotic Cell ◽

Specific Subset ◽

Suitable Target ◽

Acute Myeloid Leukemia Cells ◽

Almost All ◽

Acute Myeloid

Survivin is a 16.5 kDa protein highly expressed in centrosomes, where it controls proper sister chromatid separation. In addition to its function in mitosis, survivin is also involved in apoptosis. Overexpression of survivin in many cancer types makes it a suitable target for cancer therapy. Western blotting and confocal microscopy were used to characterize the effect of chemotherapy on acute myeloid leukemia (AML) cells. We found enhanced survivin expression in a panel of AML cell lines treated with cytarabine (Ara-C), which is part of a first-line induction regimen for AML therapy. Simultaneously, Ara-C caused growth arrest and depletion of the mitotic cell fraction. Subsequently, the effect of a second component of standard therapy protocol, idarubicin, and of a known survivin inhibitor, YM-155, on cell viability and survivin expression and localization in AML cells was investigated. Idarubicin reversed Ara-C-induced survivin upregulation in the majority of AML cell lines. YM-155 caused survivin deregulation together with a viability decrease in cells resistant to idarubicin treatment, suggesting that YM-155 might be efficient in a specific subset of AML patients. Expression levels of other apoptosis-related proteins, in particular X-linked inhibitor of apoptosis (XIAP), Mcl-1, and p53, and of the cell-cycle inhibitor p21 considerably changed in almost all cases, confirming the off-target effects of YM-155.

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