scholarly journals Cell-Type-Specific Effects of Silibinin on Vitamin D-Induced Differentiation of Acute Myeloid Leukemia Cells Are Associated with Differential Modulation of RXRα Levels

2012 ◽  
Vol 2012 ◽  
pp. 1-12 ◽  
Author(s):  
Rina Wassermann ◽  
Victoria Novik ◽  
Michael Danilenko
Keyword(s):  
Vitamin D ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
U937 Cells ◽  
Differential Modulation ◽  
Cell Type ◽  
Induced Differentiation ◽  
Response Element ◽  
Cell Type Specific ◽  
Acute Myeloid

Plant polyphenols have been shown to enhance the differentiation of acute myeloid leukemia (AML) cells induced by the hormonal form of vitamin D3 (1α,25-dihydroxyvitamin D3; 1,25D). However, how these agents modulate 1,25D effects in different subtypes of AML cells remains poorly understood. Here, we show that both carnosic acid (CA) and silibinin (SIL) synergistically enhancd 1,25D-induced differentiation of myeloblastic HL60 cells. However, in promonocytic U937 cells, only CA caused potentiation while SIL attenuated 1,25D effect. The enhanced effect of 1,25D+CA was accompanied by increases in both the vitamin D receptor (VDR) and retinoid X receptor alpha (RXRα) protein levels and vitamin D response element (VDRE) transactivation in both cell lines. Similar increases were observed in HL60 cells treated with 1,25D + SIL. In U937 cells, however, SIL inhibited 1,25D-induced VDRE transactivation concomitant with downregulation of RXRα at both transcriptional and posttranscriptional levels. These inhibitory effects correlated with the inability of SIL, with or without 1,25D, to activate the Nrf2/antioxidant response element signaling pathway in U937 cells. These results suggest that opposite effects of SIL on 1,25D-induced differentiation of HL60 and U937 cells may be determined by cell-type-specific signaling and transcriptional responses to this polyphenol resulting in differential modulation of RXRα expression.

Cell Type-Specific Effects of Crizotinib in Human Acute Myeloid Leukemia with TP53 Alterations

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2563-2563
Author(s):  
Marie Lue Antony ◽  
Klara Noble-Orcutt ◽  
Oluwateniayo Ogunsan ◽  
Fiona He ◽  
Zohar Sachs
Keyword(s):  
Acute Myeloid Leukemia ◽  
Intracellular Signaling ◽  
Myeloid Leukemia ◽  
Cell Type ◽  
Mass Cytometry ◽  
Hematopoietic Stem ◽  
Specific Effects ◽  
Cell Type Specific ◽  
Acute Myeloid

Introduction: In acute myeloid leukemia (AML) standard therapies induce complete remission in 50-70% of patients, but overall two-year survival is less than 20-30% because of high relapse rates. AML with TP53 alteration is largely insensitive to chemotherapy, modern targeted agents, and hematopoietic stem cell transplantation. Mutations in TP53 are seen in approximately 20% of AML and confer a particularly poor prognosis decreasing the 1-2 year survival rates to 0-10%. Leukemia stem cells (LSCs) are defined as the cells that recapitulate the leukemia and cause relapse. Recent large-scale drug-screening efforts to map treatment vulnerabilities in AML have revealed that crizotinib can reduce the in vitro viability of unselected primary human AML bone marrow samples with TP53 alterations (Tyner et al. Nature 2018). Crizotinib is a tyrosine kinase inhibitor that targets ALK, MET, and RON and is FDA approved for treatment of lung cancer. Since LSCs are largely responsible for AML mortality, we investigated whether crizotinib targets LSCs in primary, diagnostic human AML samples with TP53 alterations. We used mass cytometry (CyTOF2) to determine the cell-type specific effects of this drug on intracellular signaling states. We profiled treated samples with a panel that includes immunophenotypic markers (including LSC markers) and intracellular signaling molecules implicated in AML pathogenesis and aggressiveness. Results: Primary human AML samples with TP53 alterations were treated with crizotinib (10 mM, n=3). There was a reduction of viability by 48 hours of in vitro treatment with crizotinib, relative to vehicle (mean 64% reduction in viability). Crizotinib abolished all colony formation in semi-solid media. Mass cytometry analysis revealed that crizotinib treatment led to a loss of cells bearing LSC-specific markers such as CD123 and TIM3. Crizotinib treatment also induced a consistent loss of phosphorylated-signaling intermediates of the STAT and p38/MAPAPKII pathways in all samples tested. Notably, crizotinib treatment also induced significant reductions in total and phosphorylated NFKB in all samples tested. Conclusions: These data reveal cell-type specific effects of crizotinib in human AML with TP53 alterations. Crizotinib induced a specific loss of LSCs and inhibition of the STAT, p38/MAPAPKII and NFKB pathways. These data suggest that crizotinib inhibition may target LSCs in AML with TP53 alterations. Figure Disclosures No relevant conflicts of interest to declare.

Repurposing Nelarabine to Induce Differentiation of Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-26
Author(s):  
Hanying Wang ◽  
Xin He ◽  
Feiteng Huang ◽  
Haojie Dong ◽  
Wei Chen ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Myeloid Differentiation ◽  
U937 Cells ◽  
Research Support ◽  
Differentiation Induction ◽  
Nsg Mice ◽  
Ras Activation ◽  
Acute Myeloid

The success of all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL) pioneered the concept of differentiation therapy. However, comparable approaches to overcome differentiation blockage for non-APL acute myeloid leukemia (AML) are hampered by lack of an effective drug discovery platform. Recently, we analyzed gene signatures of compounds (ATRA, arsenic trioxide, zalcitabine, and sodium butyrate) that trigger myeloid differentiation in the NCI-60 collection datasets and identified CD38 as the top gene upregulated by differentiation induction. We next initiated an in silico screen in the DTP database of >20,000 compounds to identify compounds that increase CD38 levels. Among those retrieved from "CellMiner" with CD38 as input, we assessed the top 193 available from NCI (r>0.6, p=0) for effects on differentiation utilizing a conditional murine myeloid differentiation-arrest model overexpressing estrogen receptor-HoxA9 (ER-HoxA9) fusion proteins (Cell, 2016). We identified NSC755985 (Nelarabine, NEL) in that screen. NEL is an orphan drug approved to treat relapsed or refractory T-cell acute lymphoblastic leukemia (ALL). NEL at clinically achievable doses (Cmax: 6.73 μM~ 26.91 μM, at a proposed adult dosing schedule of 1,500 mg/m2/day) markedly induced primary AML cell differentiation and death while sparing normal hematopoietic cells (AML vs normal, IC50: 14.7 ± 4.3 μM, n=7, vs 45.3 ± 1.3 μM, n=3), suggesting a therapeutic window in AML. Ex vivo NEL treatment compromised BM engraftment of CD34+ cells from one primary AML specimen in immunodeficient NSG mice at 12 weeks post-transplant (human CD45+ cells in BM: NEL 0.73% vs vehicle 33.42%, n=6/group, p<0.01). NEL administration in vivo (130 mg/kg/day, i.v. for 5 consecutive days) reduced leukemic burden of NSG mice xenografted with luciferase-expressing U937 cells (Radiance: NEL 2.83×107 vs vehicle 1.65×108 photons/s/cm2, n=9/group,p<0.01) and extended mouse survival. Transcriptome analyses in U397 cells and primary AML specimens revealed that NEL treatment upregulated RAS-related pathways. NEL-elicited RAS activation was confirmed by pull-down assay using a GST-Raf1-RBD affinity probe, followed by blotting with a pan-RAS antibody. We performed functional analysis by infecting ER-HoxA9 cells with lentiviral vector expressing oncogenic RAS and observed enhanced myeloid differentiation, as evidenced by increased CD11b/GFP levels relative to MOCK-infected controls. Given that NEL's active metabolite Ara-GTP perturbs guanine nucleotide metabolism, we asked if NEL-evoked RAS activation was associated with accrual of intracellular GTP. HPLC/MS analyses of U937 cells showed that NEL treatment resulted in a marked increase in GTP (approximately 5-fold higher than baseline at sub-millimolar levels) which was secondary to Ara-GTP. Importantly, either electroporation of GTP into U937 cells or indirect introduction of GTP by addition of guanine utilizing purine salvage pathways activated RAS and recapitulated differentiation induction phenotypes. To determine whether AML cells with higher RAS activity exhibited greater NEL sensitivity, we pretreated U937 cells with a RAS agonist KRA-553 or ectopically expressed RAS mutants and observed enhanced NEL inhibitory effects in both cases. We also observed enhanced vulnerability to NEL treatment in MLL-AF9 transformed murine hematopoietic cells from KrasLox-Stop-Lox (LSL) G12D/+/Vav-Cre mice (Blood, 2009) versus Cre+ counterparts. Relevant to AML line THP-1 which is poorly responsive to NEL (IC50>100 µM), we observed extremely low levels of Ara-GTP, no GTP increase or RAS hyperactivation after NEL treatment; Ara-GTP is inactivated by SAM domain and HD domain-containing protein 1 (SAMHD1), a dNTP hydrolase, whose high expression reportedly underlies NEL resistance in T-ALL. Indeed, SAMHD1 deletion remarkably increased RAS activity in THP-1 cells treated with NEL, thereby fully reversing NEL resistance. Our study provides a preclinical basis for testing NEL efficacy in a large cohort of AML patients, given that RAS activity is generally high in AML, or even against other malignancies harboring RAS mutations, which are considered "undruggable". Additionally, further study to test whether SAMHD1 inhibition enhances NEL efficacy against RAS active cancers is warranted. Disclosures Marcucci: Pfizer: Other: Research Support (Investigation Initiated Clinical Trial); Novartis: Speakers Bureau; Takeda: Other: Research Support (Investigation Initiated Clinical Trial); Iaso Bio: Membership on an entity's Board of Directors or advisory committees; Merck: Other: Research Support (Investigation Initiated Clinical Trial); Abbvie: Speakers Bureau. Sykes:Clear Creek Bio: Current equity holder in private company, Other: co-founder.

Targeted Gene Therapy Vectors for Acute Myeloid Leukemia Cells Selected from Random AAV-Displayed Peptide Libraries.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3176-3176
Author(s):  
Martin Trepel ◽  
Mi-Kyung Lee ◽  
Felix Kaul ◽  
Jurgen Kleinschmidt
Keyword(s):  
Gene Therapy ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Cell Types ◽  
Peptide Libraries ◽  
Leukemia Cells ◽  
Cell Type ◽  
Gene Therapy Vectors ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract For acute myeloid leukemia, gene therapy may be a valuable tool to treat patients refractory to conventional chemotherapy regimens. However, there are no vectors available that sufficiently and specifically transduce this cell type. Cell type specific receptors can be used to target gene therapy vectors to the cells of interest (Trepel et al., Hum. Gene Ther., 2001). We have developed a screening system based on random peptide libraries displayed on adeno-associated virus type 2 (AAV) (Muller et al., Nat. Biotechnology, 2003). Such libraries consist of up to 108 different viruses that display a targeting peptide with random sequence in a capsid region that mediates binding of the virus particle to cell surface receptors. Targeted AAV gene therapy vectors that specifically transduce the cell types of interest can be selected from such a library. Here we report the selection of a random AAV display peptide library on a panel of acute myeloid leukemia cell lines (Kasumi-1, U937, HL 60). An X7 (X = any amino acid) oligonucleotide insert was cloned in to the AAV-library plasmid followed by the production of AAV library transfer shuttle AAV. To produce the final library, AAV producer cells were infected with shuttle AAV at an MOI of 1 to ensure encoding of displayed peptides by the packaged DNA. The target cells were then infected with the AAV library. Bound particles were internalized and amplified either by superfinfection of the cells with wild type adenovirus type 5 or by a high-sensitivity PCR-system of the random oligonucleotide followed by cloning of the insert and making a preselected library as above. Amplified clones were recovered and subjected to two more rounds of selection. Enriched peptide inserts were analyzed indirectly by DNA sequencing of recovered clones. After three rounds of selection, enrichment of a peptide motif within the selected AAV capsids was observed. The selected viruses were used for production of recombinant AAV vectors harboring a GFP reporter gene. Such recombinant targeted vectors transduced the target leukemia cells they have been selected on (e.g., Kasumi-1 cells) up to 60 times more efficient compared to a random clone of the initial, unselected library. We conclude that our novel targeting strategy can be applied to a great variety of cell types. The clones selected here may be used as valuable tools to target therapeutic genes to acute myeloid leukemia cells.

A Novel Path for ATRA Differentiation Therapy in Acute Myeloid Leukemia with Isocitrate Dehydrogenase Mutations

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3727-3727
Author(s):  
Jean-Emmanuel Sarry ◽  
Helena Boutzen ◽  
Christian Récher
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Granulocytic Differentiation ◽  
Induced Differentiation ◽  
Idh1 R132h ◽  
Recurrent Mutations ◽  
Idh Mutations ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is characterized by accumulation of malignant blasts with impaired differentiation programs due to recurrent mutations, among which IDH mutations occur in 15% of AML patients. These mutations lead to a block in erythroid commitment while they may also bias hematopoietic differentiation to myeloid lineage. Interestingly, Lyn tyrosine kinase is required for erythroid differentiation and we have observed a reduction of Lyn expression in the presence of IDH1-R132H mutation. It is also a negative regulator of ATRA-induced granulocytic differentiation. Accordingly, we hypothesized that IDH mutations may sensitize AML cells to ATRA-induced differentiation. Here, we report that clinically achievable doses of ATRA are sufficient to trigger differentiation specifically on AML cell lines, primary patient samples and xenograft mice models carrying IDH1 mutation as observed by an increase in CD11b expression, granulocytic enzyme activity and morphologic changes in May-Grunwald-Giemsa staining. We also showed that ATRA-induced terminal granulocytic differentiation increases apoptosis while decreases proliferation and colony formation specifically in IDH1 mutant cells. Moreover, inhibition of IDH1-R132H activity reduced ATRA-sensitivity while increasing expression of IDH mutation correlated with highest ATRA sensitivity. Furthermore, treatment with a cell-permeable form of the oncometabolite specifically produced by the mutant (eg. 2-HydroxyGlutarate) sensitized AML cells to ATRA-induced differentiation. Finally, because ATRA-induced differentiation triggers a transient increase of Lyn activation, its association with Lyn inhibitors synergistically increased ATRA-induced differentiation of IDH mutant blasts. In summary, our results showed that IDH mutations by producing 2-HG sensitized leukemic blasts to ATRA and that this synergizes with Lyn inhibition. Since 2HG concentration reaches millimolar in AML patient serum and is 100-fold higher in IDH mutated patients than in non-mutated ones, we would predict a strong efficacy and specificity of ATRA. Furthermore, as IDH mutations are systematically conserved at relapse, this therapeutic strategy might be promising to achieve a long-term remission specifically for this AML patient subgroup. Disclosures No relevant conflicts of interest to declare.

C/EBPα and MiR-182 Generate a Negative Feedback Loop Which Is Dysregulated in Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 776-776
Author(s):  
Alexander Arthur Wurm ◽  
Dennis Gerloff ◽  
Daniela Braeuer-Hartmann ◽  
Christiane Katzerke ◽  
Jens-Uwe Hartmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Feedback Loop ◽  
Mononuclear Cells ◽  
Myeloid Differentiation ◽  
Negative Feedback Loop ◽  
U937 Cells ◽  
Leukemia Development ◽  
Acute Myeloid

Abstract The transcription factor CCAAT enhancer binding protein alpha (C/EBPα) is a master regulator of granulopoiesis and is silenced in approximately 50% of all acute myeloid leukemia (AML) cases. There are several mechanisms known how C/EBPα is inactivated in AML, including promoter hypermethylation, posttranslational modifications and mutations in the ORF of the CEBPA gene. MicroRNAs, a class of small non-coding RNAs, were identified as important regulators of normal hematopoiesis and leukemia development. We have already shown that microRNAs, such as miR-223, miR-34a and miR-30c, are essential elements in C/EBPα triggered granulocytic differentiation. But to our knowledge nothing is known about inactivation of C/EBPα by microRNAs in acute myeloid leukemia. In this study, we identified a novel network between C/EBPα and miR-182. In a next generation sequencing approach based on inducible K562-C/EBPα-ER cell line, we found miR-182 strongly downregulated by wildtype C/EBPα. We could further demonstrate an inverse correlation between C/EBPα protein amount and miR-182 expression level in several in vitro systems, including leukemic cell lines and G-CSF treated primary human CD34+progenitor cells. Additionally, C/EBPα and miR-182 showed reciprocal expression in sorted murine bone marrow subpopulations in vivo. To discover the mechanism how miR-182 is blocked by C/EBPα, we analyzed the minimal promoter region of miR-182 and performed chromatin immunoprecipitation (ChIP). Here, we could demonstrate a strong binding of C/EBPα to the miR-182 promoter, particularly to a conserved E2F binding site. Because E2F is a well known inhibitor of C/EBPα function, we tested whether E2F also effects miR-182 expression. An overexpression of E2F1 in U937 cells leads to an elevated miR-182 expression level. In addition, we measured the expression of miR-182 in bone marrow from AML patients regarding to their CEBPA mutation status. We could show that only patients with mutations in the C-terminal region of C/EBPα showed elevated miR-182 expression, while patients with N-terminal CEBPA mutations revealed no abnormal miR-182 expression compared to healthy donors or AML patients with no CEBPA mutation. The C-terminal domain of C/EBPα is necessary for E2F inhibition. These findings illustrate the importance of C/EBPα-E2F interaction during miR-182 regulation. Next, we found a highly conserved binding site of miR-182 in the 3’UTR of CEBPA itself, suggesting a possible negative feedback loop. To test this, we performed overexpression of miR-182 in U937 cells, umbilical cord blood mononuclear cells (UCB-MNCs) and primary blasts from AML patients. Here, we observed a strong reduction of C/EBPα protein after miR-182 overexpression in all cell types. Furthermore, we could demonstrate a direct binding of miR-182 to the 3’UTR of CEBPA via luciferase activity assay. Finally, we were interested in the functional impact of miR-182 in myeloid differentiation and leukemia development. We showed that enforced expression of miR-182 in U937 cells reduced the percentage of Mac-1 positive myeloid cells after treatment with all-trans retinoid acid (ATRA). Additionally, lentiviral overexpression of miR-182 induces a block of differentiation and hyperproliferation in G-CSF treated 32D cells and an enhanced replating capacity of primary mouse bone marrow mononuclear cells. Taken together, we identified miR-182 as novel oncogenic microRNA that directly blocks C/EBPα during myeloid differentiation and leukemia development. Thus, our data display a potential new strategy for therapeutics in C/EBPα dysregulated AML. Disclosures No relevant conflicts of interest to declare.

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