scholarly journals A Novel Chemical Compound Inhibiting Hematopoietic Cell Kinase (iHCK) Has a Synergic Effect with Azacytidine (Aza) or Cytarabine (Ara-C) for Acute Myeloid Leukemia Treatment

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4650-4650
Author(s):  
Fernanda Marconi Roversi ◽  
Maura Lima Pereira Bueno ◽  
Cristiane Okuda Torello ◽  
Fernanda I Della Via ◽  
Renata Giardini Rosa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Hematopoietic Cell Kinase ◽  
Leukemia Cell Lines ◽  
Cd34 Cells ◽  
Acute Myeloid

Introduction. Hematopoietic cell kinase (HCK) belongs to the Src kinase family (SFK) involved in the oncogenic process and hematological malignancy. Some SFK inhibitors are currently under investigation in clinical trials for leukemia after demonstrating efficacy in patients with solid tumors. We have previously reported that HCK is overexpressed in leukemic cells and its inhibition by lentivirus resulted in reduction of cell growth and increased cell death (Roversi et al. BBA Mol Basis Dis. 2017, 1863(2):450-61). In light of the genomic and molecular diversity of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), the development of chemical compounds specific for new molecular targets is currently an important subject. Aims. To investigate the in vitro and in vivo effects of a new chemical compound targeting HCK inhibition (iHCK), alone or in combination with the most used drugs for treatment of MDS and AML (Azacytidine - Aza - or Cytarabine - Ara-C). Methods. After iHCK development, we tested its activity alone or in combination with Aza or Ara-C in CD34+ cells isolated from AML patients (n=5) as well as in a panel of myeloid leukemia cell lines (KG1, HL-60, HEL and K562). Additionally, we tested the iHCK in normal and malignant cells cultured in a 3D bioscaffold obtained by decellularization of bovine bone marrow (Bianco et al. Biomat Sci 2019, 7(4):1516-28), in order to mimic the bone marrow niche. After informed written consent and approval of the Ethical Committee of University of Campinas (CAAE 1000.0.146.00-11), in accordance to the Helsinki Declaration, CD34+ cells were isolated from bone marrows of healthy donors (HD), MDS and AML patients and were treated with iHCK or vehicle (DMSO) in liquid culture, for three days. Meanwhile, HS-5 mesenchymal cells were cultured into the 3D bioscaffold. iHCK or vehicle treated CD34+ cells were introduced into the 3D bioscaffold containing HS-5 and evaluated after 7 and 14 days, by light microscopy (hematoxilin and eosin regular staining) and immunohistochemistry (expression of CD34 and CD90 antigens). NOD.CB17-Prkdcscid/J mice received 2 Gy irradiation followed by transplantation with caudal intravenous injection of leukemia cells obtained from hCG-PML-RARα transgenic mice. After acute promyelocytic leukemia (APL) establishment, animals were treated or not with intraperitoneally iHCK and peripheral blood was collected for hematological analysis and protein was extracted from spleen and bone marrows for Western Blot analysis. ANOVA and Student's T-Test were used. Results.In leukemia cell lines and primary cells, the combinatory treatment of iHCK and Cytarabine (1μM) or 5-Azacitidine (1μM) demonstrated synergistic effects, compared to either drug alone, on the reduction of growth and induction of cell death (P<0.001; Figure 1). Further, Western blot revealed increased BAX expression and decreased BCL-XL expression. Moreover, iHCK treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to severe reduction of ERK, AKT and p70S6 phosphorylation. Treatment with iHCK reduced CD34+ MDS and AML cells proliferation cultured into the 3D bioscaffold but had no effect upon normal CD34+cells. In vivo analysis showed that APL mice treated with iHCK (5μM) for 48h had reduced leukocyte number compared to APL mice treated with vehicle (13.2±1.1 vs 49.4±18.8; P<0.001). No alterations in hemoglobin levels and platelet were found. Likewise, the in vivo iHCK (2.5μM, 5.0μM or 10.0μM) treatment decreased the phosphorylation of ERK, AKT and P70S6K proteins of leukemic cells (Figure 2). Conclusion.The iHCK pharmacological inhibitor has an antiproliferative activity in leukemic cells without altering cell death and survival rate of normal cells, demonstrating on-target malignant cell killing activity as a single agent or in combination with Azacytidine (Aza) or Cytarabine (Ara-C). Disclosures No relevant conflicts of interest to declare.

Repression of Mir-495, a Microrna Associated with Favorable Outcome of Acute Myeloid Leukemia Patients, Is Required for the MLL-Associated Leukemogenesis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3462-3462
Author(s):  
Xi Jiang ◽  
Hao Huang ◽  
Zejuan Li ◽  
Yuanyuan Li ◽  
Ping Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Mouse Bone Marrow ◽  
Human Leukemia ◽  
Leukemia Cell Lines ◽  
Acute Myeloid

Abstract Abstract 3462 Acute myeloid leukemia (AML) bearing MLL (mixed lineage leukemia) translocations are associated with poor survival, and only fewer than 50% of the patients survive longer than 5 years. Thus, an improved strategy leading to a higher cure rate is urgently needed to treat MLL-associated AML. MicroRNAs (miRNAs), a class of small non-coding RNAs, have been postulated to be important gene expression regulators in all biology including human leukemia. Through large-scale, genome-wide miRNA expression profiling assays, we determined that miR-495 is significantly down-regulated in the majority of human AML samples, particularly, in those with MLL rearrangements. More interestingly, through correlating the expression signature of miR-495 with clinical outcome of AML patients, we revealed that a low expression level of miR-495 is a predictor of poor prognosis in most AML patients. Our further qPCR assays confirmed that the expression of miR-495 is even more significantly downregulated in MLL-rearranged AML primary patient samples and cell lines. Through in vitro colony-forming/replating assays and in vivo bone marrow transplantation studies, we found that forced expression of miR-495 significantly inhibits the capacity of the MLL-AF9 fusion gene to support colony formation in mouse bone marrow progenitor cells in vitro and to induce leukemia in vivo. In leukemia cell lines, overexpression of miR-495 greatly inhibits the viability of the cells, while increasing apoptosis. Furthermore, by using 3 algorithms for miR-495 3'UTR binding sites, we identified several well-known MLL leukemia-related genes, e. g. BMI1, MEF2C, BID and MEIS1, as potential targets of miR-495. Results of qPCR revealed that forced expression of miR-495 significantly inhibits the expression levels of these genes in leukemia cell lines, mouse bone marrow progenitor cells, as well as mouse peripheral blood cells with MLL fusion genes. Therefore we hypothesize that miR-495 may function as a tumor suppressor in AML with MLL rearrangements by targeting essential tumor-related genes. Further studies will focus on: 1) effects of miR-495 on the functions of target genes studied in vitro and in vivo; 2) the epigenetic mechanisms and the signaling pathways involved in regulating the expression level of miR-495 in human leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals E4F1 deficiency results in oxidative stress–mediated cell death of leukemic cells

2011 ◽  
Vol 208 (7) ◽  
pp. 1403-1417 ◽  
Author(s):  
Elodie Hatchi ◽  
Genevieve Rodier ◽  
Matthieu Lacroix ◽  
Julie Caramel ◽  
Olivier Kirsh ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Tumor Regression ◽  
Fetal Liver ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Viral Oncoprotein ◽  
Mitochondrial Defects

The multifunctional E4F1 protein was originally discovered as a target of the E1A viral oncoprotein. Growing evidence indicates that E4F1 is involved in key signaling pathways commonly deregulated during cell transformation. In this study, we investigate the influence of E4F1 on tumorigenesis. Wild-type mice injected with fetal liver cells from mice lacking CDKN2A, the gene encoding Ink4a/Arf, developed histiocytic sarcomas (HSs), a tumor originating from the monocytic/macrophagic lineage. Cre-mediated deletion of E4F1 resulted in the death of HS cells and tumor regression in vivo and extended the lifespan of recipient animals. In murine and human HS cell lines, E4F1 inactivation resulted in mitochondrial defects and increased production of reactive oxygen species (ROS) that triggered massive cell death. Notably, these defects of E4F1 depletion were observed in HS cells but not healthy primary macrophages. Short hairpin RNA–mediated depletion of E4F1 induced mitochondrial defects and ROS-mediated death in several human myeloid leukemia cell lines. E4F1 protein is overexpressed in a large subset of human acute myeloid leukemia samples. Together, these data reveal a role for E4F1 in the survival of myeloid leukemic cells and support the notion that targeting E4F1 activities might have therapeutic interest.

In vivo administration of vascular endothelial growth factor (VEGF) and its antagonist, soluble neuropilin-1, predicts a role of VEGF in the progression of acute myeloid leukemia in vivo

Blood ◽  
2002 ◽  
Vol 100 (13) ◽  
pp. 4622-4628 ◽  
Author(s):  
Gunter Schuch ◽  
Marcelle Machluf ◽  
Georg Bartsch ◽  
Masashi Nomi ◽  
Henri Richard ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Vascular Endothelial Growth Factor ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Vascular Endothelial ◽  
Neuropilin 1 ◽  
Endothelial Growth Factor ◽  
Acute Myeloid

Recent findings implied that the progression of hematologic malignancies, like that of solid tumors, is dependent on neovascularization. Recent studies on patients with acute myeloid leukemia (AML) showed increased levels of leukocyte-associated vascular endothelial growth factor (VEGF) and neovascularization of the bone marrow. Murine (32D, M1) and human (HEL, U937, and UKE-1) leukemic cell lines and freshly isolated leukemic cells were analyzed for the expression of VEGF and VEGF receptor mRNA. The expression of VEGF and VEGF receptors KDR and neuropilin-1 (NRP-1) was detected in these cells. In a murine chloroma model, delivery of VEGF165using microencapsulation technology resulted in enhanced tumor growth and vascularization, whereas treatment with a VEGF antagonist soluble NRP-1 (sNRP-1) inhibited tumor angiogenesis and growth. In a systemic leukemia model, survival of mice injected with adenovirus (Ad) encoding for Fc-sNRP-1 (sNRP-1 dimer) was significantly prolonged as compared with mice injected with Ad-LacZ. Further analyses showed a reduction in circulating leukemic cells and infiltration of liver and spleen as well as bone marrow neovascularization and cellularity. Taken together, these results demonstrate that angiogenic factors such as VEGF promote AML progression in vivo. The use of VEGF antagonists as an antiangiogenesis approach offers a potential treatment for AML. Finally, our novel in vivo drug delivery model may be useful for testing the activities of other peptide antiangiogenic factors.

In Vitro and In Vivo Monitoring of Valproic Acid Effects on Gene Expression Signatures in Adult Acute Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2605-2605
Author(s):  
Lars Bullinger ◽  
Konstanze Dohner ◽  
Richard F. Schlenk ◽  
Frank G. Rucker ◽  
Jonathan R. Pollack ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Serum Levels ◽  
Leukemia Cell Lines ◽  
Acute Myeloid ◽  
Myeloid Leukemia Cell

Abstract Inhibitors of histone deacetylases (HDACIs) like valproic acid (VPA) display activity in murine leukemia models, and induce tumor-selective cytoxicity against blasts from patients with acute myeloid leukemia (AML). However, despite of the existing knowledge of the potential function of HDACIs, there remain many unsolved questions especially regarding the factors that determine whether a cancer cell undergoes cell cycle arrest, differentiation, or death in response to HDACIs. Furthermore, there is still limited data on HDACIs effects in vivo, as well as HDACIs function in combination with standard induction chemotherapy, as most studies evaluated HDACIs as single agent in vitro. Thus, our first goal was to determine a VPA response signature in different myeloid leukemia cell lines in vitro, followed by an in vivo analysis of VPA effects in blasts from adult de novo AML patients entered within two randomized multicenter treatment trials of the German-Austrian AML Study Group. To define an VPA in vitro “response signature” we profiled gene expression in myeloid leukemia cell lines (HL-60, NB-4, HEL-1, CMK and K-562) following 48 hours of VPA treatment by using DNA Microarray technology. In accordance with previous studies in vitro VPA treatment of myeloid cell lines induced the expression of the cyclin-dependent kinase inhibitors CDKN1A and CDKN2D coding for p21 and p19, respectively. Supervised analyses revealed many genes known to be associated with a G1 arrest. In all cell lines except for CMK we examined an up-regulation of TNFSF10 coding for TRAIL, as well as differential regulation of other genes involved in apoptosis. Furthermore, gene set enrichment analyses showed a significant down-regulation of genes involved in DNA metabolism and DNA repair. Next, we evaluated the VPA effects on gene expression in AML samples collected within the AMLSG 07-04 trial for younger (age<60yrs) and within the AMLSG 06-04 trial for older adults (age>60yrs), in which patients are randomized to receive standard induction chemotherapy (idarubicine, cytarabine, and etoposide = ICE) with or without concomitant VPA. We profiled gene expression in diagnostic AML blasts and following 48 hours of treatment with ICE or ICE/VPA. First results from our ongoing analysis of in vivo VPA treated samples are in accordance with our cell line experiments as e.g. we also see an induction of CDKN1A expression. However, the picture observed is less homogenous as concomitant administration of ICE, as well as other factors, like e.g. VPA serum levels, might substantially influence the in vivo VPA response. Nevertheless, our data are likely to provide new insights into the VPA effect in vivo, and this study may proof to be useful to predict AML patients likely to benefit from VPA treatment. To achieve this goal, we are currently analyzing additional samples, and we are planning to correlate gene expression findings with histone acetylation status, VPA serum levels, cytogenetic, and molecular genetic data.

Evidence for CD34+ Hematopoietic Progenitor Cell Involvement in Acute Myeloid Leukemia with NPM1 Gene Mutation: Implications for the Cell of Origin

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 307-307 ◽  
Author(s):  
Maria Paola Martelli ◽  
Valentina Pettirossi ◽  
Elisabetta Bonifacio ◽  
Federica Mezzasoma ◽  
Nicla Manes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Hox Genes ◽  
Leukemic Cells ◽  
Mutant Protein ◽  
Cell Of Origin ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Acute Myeloid

Abstract Acute myeloid leukemia expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM2005;352:254–266] is a new entity of WHO classification that shows distinctive biological and clinical features, including a unique molecular signature characterized by downregulation of CD34 and upregulation of most HOX genes [Falini B et al, Blood2007;109:874–885]. Involvement of HOX genes in the maintenance of the stem-cell phenotype strongly suggest that AML with mutated NPM1 originates from a multipotent hematopoietic progenitor (HSC). This view is also supported by immunohistological findings showing that AML with mutated NPM1 frequently displays multilineage involvement [Pasqualucci L et al, Blood2006;108:4146–4155]. On the other hand, the frequent negativity of NPMc+ AML for the HSC-associated antigen CD34 raises the question of whether the mutation event occurs in a CD34-negative HSC (these cells have been identified in mice) or whether a minimal pool of CD34-positive NPM1-mutated leukemic cells does exist. Currently, the hierarchical level of stem cell involvement in NPMc+ AML is unknown. To address this issue, we purified CD34+ cells from NPMc+ AML patients and detected NPM1 mutant protein in the sorted population by Western blot with anti-NPM mutant specific antibodies [Martelli MP et al, Leukemia 2008] (Figure 1A). We investigated 6 NPMc+ AML patients presenting at diagnosis with 0.12%, 0.14%, 0.38%, 5%, 22%, and 28% of CD34+ cells in the peripheral blood. In all cases, CD34+ fractions (purity &gt;90%) harboured NPM1 mutant protein, indicating they belong to the leukemic clone (Figure 1B). The percentage of most undifferentiated CD34+/CD38− cells in the CD34+ fractions ranged from 5 to 97%. Notably, in at least one case, all CD34+ NPM1-mutated leukemic cells were CD38−negative. Moreover in all cases, CD34+ NPM1-mutated leukemic cells appeared to express CD123 (IL-3 receptor), considered a marker of the leukemic stem cell and target of potential therapy. Double staining of bone marrow biopsies with anti-CD34 and anti-NPM antibodies revealed that the rare CD34+ cells expressed NPM1 aberrantly in the cytoplasm. Inoculation of CD34+ NPM1-mutated AML cells into sublethally irradiated NOD/SCID mice resulted into leukemia engrafment in various body sites, especially bone marrow, spleen, lung and liver. Preliminary results showed that CD34+ leukemic cells reacquired the same leukemic phenotype as the original patient’s, including CD34-negativity of the leukemic bulk in spite of any lack of differentiation. This finding suggests that NPM1 mutant protein may be involved in downregulation of CD34 antigen, while keeping a gene expression profile typical of the hematopoietic stem cell. These findings suggest the CD34+ fraction contains the SCID-leukemia initiating cells (SL-IC) and point to CD34+/CD38− HSC as the cell of origin of AML with mutated NPM1. Figure Figure

Disulfiram, an clinically used anti-alcoholism drug has the potential to target acute myeloid leukemia cells in a copper-dependent manner in vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5040-5040
Author(s):  
Bing Xu ◽  
Rongwei Li ◽  
Huijuan Dong ◽  
Feili Chen ◽  
Yuejian Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Specific Antigen ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Control Group ◽  
Acute Myeloid

Abstract Background Disulfiram(DS), an old drug clinically used for alcoholism, was reported to have antitumor effects, recent studies have found that Copper(Cu) can significantly enhance the DS-induced cell death in vitro in a variety of tumor cells. Our previous studies also demonstrated that disulfiram/copper (DS/Cu) couldtarget human leukemia cell lines(like KG1α,Molt4) through the activation of JNK, in vitro. However, there is few report about the ability of DS/Cu in killing cancer cells in vivo. Aims This study aims to explore the effect of DS/Cu on acute myeloid leukemia cell line KG1αin vivo and clarify the underlining mechanism. Methods 6-8 week old female NOD/SCID mice were sublethally irradiated with 2Gy X-ray the day before transplantation, followed by intravenous injection of KG1α cells (1×107 cells) suspended in 0.2 mL of PBS. 5 weeks after transplantation mice were randomly divided into three treatment groups: vehicle (0.9% saline), a combination of DS and Cu daily for 2 weeks, Ara-C alone twice before killing. Mice were sacrificed after 2 weeks treatment with tissues of spleen, liver, bone marrow being observed using histopathology method to detect the invasion of leukemia. The DS/Cu-induced p-c-jun activation was also examined by western blot using tissues of spleen, liver, bone marrow. Statistical analysis was carried out with one-way ANOVA to assess statistical significance (*p < 0.05). Results 4 weeks after transplantation, mice were dispirited with low appetite, down-bent gait, wrinkled fur, slow move, just like suffered from leukemia. What’s more, immature blasts like morphology similar to KG1α were found in the peripheral blood of the mice(11%±3.41). All the mice were sacrificed after 2 weeks treatment, mice in control group were observed with slightly larger spleen and liver with the morphology of invasion of leukemia such as a granular appearance than the other two groups. Histopathology examination showed that leukemia cells infiltrate liver, spleen and bone marrow, and the immunohistochemistry examination found that the leukemia cells in spleen, liver and bone marrow expressed human specific antigen CD45 with the highest expression level in the control group. Moreover, solid tumor could be observed in the peritoneal cavity of two mice in the control group with expression of human specific antigen CD45detected by immunohistochemistry examination. Western blot in this study showed DS/Cu complex induced phosphorylation of c-Jun expression in the spleen, liver and bone marrow. Conclusion DS/Cu complex could effectively target the acute myeloid leukemia cells in the acute leukemia NOD/SCID mice while inhibiting the invasion of leukemia to some extent, and the activation of JNK might play a functional role in DS/Cu mediated antileukemic effects. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Impact of Bone Marrow miR-21 Expression on Acute Myeloid Leukemia T Lymphocyte Fragility and Dysfunction

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2053
Author(s):  
Douâa Moussa Agha ◽  
Redouane Rouas ◽  
Mehdi Najar ◽  
Fatima Bouhtit ◽  
Hussein Fayyad-Kazan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
T Lymphocytes ◽  
T Lymphocyte ◽  
Myeloid Leukemia ◽  
Myeloid Cell ◽  
Leukemic Cells ◽  
Hematopoietic Malignancy ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy in which antitumor immunity is impaired. The therapeutic management of AML requires understanding the mechanisms involved in the fragility and immune dysfunction of AML T lymphocytes. Methods: In this study, T lymphocytes from healthy donors (HD) and AML patients were used. Extracellular vesicles (EVs) from leukemic cells were screened for their microRNA content and impact on T lymphocytes. Flow cytometry, transcriptomic as well as lentiviral transduction techniques were used to carry out the research. Results: We observed increased cell death of T lymphocytes from AML patients. EVs from leukemia myeloid cell lines harbored several miRNAs, including miR-21, and were able to induce T lymphocyte death. Compared to that in HD, miR-21 was overexpressed in both the bone marrow fluid and infiltrating T lymphocytes of AML patients. MiR-21 induces T lymphocyte cell death by upregulating proapoptotic gene expression. It also increases the immunosuppressive profile of T lymphocytes by upregulating the IL13, IL4, IL10, and FoxP3 genes. Conclusions: Our results demonstrate that miR-21 plays a significant role in AML T lymphocyte dysfunction and apoptosis. Targeting miR-21 may be a novel approach to restore the efficacy of the immune response against AML.

scholarly journals Thyrointegrin αVβ3 Antagonist: Implications in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4434-4434
Author(s):  
Noureldien Darwish ◽  
Gennadi V. Glinsky ◽  
Shaker A Mousa
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Anticancer Activity ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Biological Functions ◽  
Regulated Gene Expression ◽  
Acute Myeloid

Abstract Leukemic cells are able to receive and send several signals within bone marrow niche that play an important role in their survival. One of the important crosstalk is the interaction between the bone marrow microenvironment proteins (vitronectin, fibronectin, fibrinogen, and ostepontin) and thyrointegrin αVβ3 on leukemic cells, generating ligand-specific outside-in signals that are relevant to a variety of cell functions, including gene transcription, cell division, cell attachment, and motility Our previous experiment using in vivo AML animal models with primary AML cells and cell lines have shown significant reduction of leukemic cell burden 74% and &gt;95% (P&lt;0.0001), respectively, after daily subcutaneous treatment with thyrointegrin αvβ3 antagonist fb-PMT (Ki 0.23 nM) at 3 and 10 mg/kg, for 3-4 weeks. In this study we focused on evaluations of the molecular effects of fb-PMT in leukemic cells. Acute myeloid leukemia cell lines (K562-Luc and KG1a cells) were cultured in 50 cm² cell culture flasks with 10 mL phenol red free RPMI media containing 10% fetal bovine albumin. The leukemic cells were treated (at 50% confluence) with 30 µM fb-PMT for 48 hours. Total RNA was immediately isolated from harvested cells using Triazole and used for microarray analysis. Overall, there were 370 significantly down-regulated gene expression records and 273 significantly up-regulated gene expression records, expression of which were changed at least 1.5-fold in fb-PMT-treated human leukemic cells. Significant examples of the fb-PMT-induced gene expression signatures (GES) of pathway's interference include SNAI, MYC, HIF1A, TWIST1, and TFAP2C (P&lt;0.05). Notably, inference of potential contribution to the fb-PMT anticancer activity of the interference with these pathways seems highly congruent with their known biological functions such as cell cycle control (MYC), survival and maintenance of stem cells (HIF1A, TFAP2C), and essential features of the malignant phenotype (TWIST1, SNAI) (Figure 1). Consistently, examples of the fb-PMT-induced GES of transcriptional pathway's activation include RB1, IRF9, MAML1, RAP1A, and GATA4 pathways (P&lt;0.05), known biological functions of which appear highly consistent with the hypothesis that activation of these pathways might contribute to fb-PMT anticancer activity. Finally, we found that fb-PMT interfered with estrogen signaling in human AML cells. The fb-PMT was associated with decreased phosphorylation and nuclear enrichment of Erα (Figure 1). Collectively, our in vivo study and genomic data have shown the key role thyrointegrin αvβ3 in leukemogenesis. The thyrointegrin αvβ3 antagonist fb-PMT demonstrated potent anticancer actions on human AML through the molecular interference mechanism with multiple signaling pathways supporting growth and survival of leukemic cells Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of CHD4 As a Potential Therapeutic Target of Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1648-1648 ◽  
Author(s):  
Yaser Heshmati ◽  
Gözde Turköz ◽  
Aditya Harisankar ◽  
Sten Linnarsson ◽  
Marios Dimitriou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Loss Of Function ◽  
Potential Therapeutic Target ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate. The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML. In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs. The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells. To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells. Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo. Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation. Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. Disclosures No relevant conflicts of interest to declare.

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