scholarly journals NFATC2 regulates Targets of MYC Signaling in MLL-AF9 AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3301-3301
Author(s):  
Shaun David Patterson ◽  
Matthew E Massett ◽  
Helen Wheadon ◽  
Xu Huang ◽  
Heather G Jørgensen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Expression Profiles ◽  
Control Cell ◽  
Gene Set Enrichment Analysis ◽  
Transcriptional Networks ◽  
Rna Seq ◽  
Activated T Cells ◽  
Self Renewal

Abstract Background: Acute myeloid leukemia (AML) arises due to an accumulation of genetic lesions within myeloid progenitors and oncogenic transformation is often characterised by disordered transcription. Recently the histone lysine demethylase KDM4A was shown to be essential for AML blast survival and self-renewal. shRNA knockdown (KD) of KDM4A led to downregulated expression of the transcription factor NFATC2 an MLL-AF9 AML model, suggesting that it is a key target of KDM4A oncogenic function. The Nuclear Factor of Activated T Cells (NFAT) family of transcription factors control cell cycle genes and self-renewal pathways in hematopoietic tissues and are well-defined as oncogenic regulators in various malignancies. NFATs have recently been attributed roles in the development of FLT3 ITD AML and resistance to tyrosine kinase inhibitors (TKIs) in myeloid leukemias but there is little evidence detailing the role(s) of NFATC2 specifically in AML. We hypothesized that NFATc2 activity is essential for the survival of AML cells and the oncogenic transcriptional networks within these. Aims: To determine if AML cells are dependent on NFATC2 for survival and to elucidate the transcriptional and binding targets of NFATc2 in AML. Methods: NFATC2 was depleted using shRNA KD in numerous cell line models of AML and putative transcriptional targets were elucidated using RNA-seq following KD. Binding targets of NFATc2 were determined using ChIP-seq. Transcriptomic targets of NFATc2 were validated using the Fluidigm Biomark multiplex PCR system and real time quantitative PCR. Results: KD of NFATC2 significantly impaired the colony forming capacity and expansion in liquid cultures of AML cell lines from diverse (cyto)genetic backgrounds. MLL-AF9/TP53 mut THP-1 cells showed reduced entry to the S-phase of the cell cycle and downregulation of cyclin D1 following NFATC2 depletion, suggesting that NFATC2 is critical for cell cycle progression in these cells. Overexpression of human NFATC2 in THP-1 led to an increased rate of cell growth. RNA-seq analysis of THP-1 cells with NFATC2 KD revealed >20 genes with deregulated expression (FDR<0.1), which have been validated using PCR methods. Overexpression of human NFATC2 resulted in significant deregulation of 9 of these genes (FDR<0.1), defining a subset of genes which may regulate the observed phenotype. Additionally, these top genes were not all differentially regulated in other MLL-AF9 AML cell lines MOLM-13 and NOMO-1 following NFATC2 KD. Finally, in THP-1, gene set enrichment analysis (GSEA) of sequencing results revealed that targets of MYC and calmodulin kinase STK33 were enriched within the genes perturbed by NFATC2 depletion. Targets of MYC signaling were validated by PCR in THP-1 but were not found to be deregulated in MOLM-13 following NFATC2 KD. ChIP-seq analysis of NFATc2 binding in THP-1 cells showed that >30% of NFATc2 targets were at promoter regions within 5kb of the transcription start site. Motif analysis of precipitated DNA fragments discovered two novel motifs which were enriched at NFATc2 binding sites (p<0.0001). Discussion: NFATC2 was found to be essential for expansion of AML cells in various cell line models. In the MLL-AF9 driven THP-1 model a number of putative transcriptional and genomic targets were defined, which include novel targets not previously described in AML pathogenesis and targets of MYC, an established oncogenic protein in AML. The differing expression profiles observed across AML cell lines of diverse (cyto)genetic backgrounds with NFATC2 KD suggest that the regulatory targets of NFATc2 vary depending on the cellular signaling landscape. Together with the finding that NFATC2 is indispensable for AML cell survival this study has elucidated novel roles(s) for NFATC2 in AML oncogenesis. Disclosures Massett: Kymab Ltd: Current Employment. Huang: Janssen Pharmaceutical Companies of Johnson & Johnson (China): Current Employment.

scholarly journals High Mobility Group A1 Chromatin Regulators Function As Critical Drivers of Leukemogenesis in Preclinical Models of Relapsed, Pediatric B-Cell ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3946-3946
Author(s):  
Liping Li ◽  
Katharina Hayer ◽  
Lingling Xian ◽  
Li Luo ◽  
Leslie Cope ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
High Mobility ◽  
Transcriptional Networks ◽  
Rna Seq ◽  
Cycle Progression ◽  
Short Hairpin ◽  
Reh Cells

Introduction: Acute B-cell lymphoblastic leukemia (B-ALL) is the most common form of childhood leukemia and the leading cause of death in children with cancer. While therapy is often curative, about 10-15% of children will relapse with recurrent disease and abysmal outcomes. Actionable mechanisms that mediate relapse remain largely unknown. The gene encoding the High Mobility Group A1(HMGA1) chromatin regulator is overexpressed in diverse malignancies where high levels portend poor outcomes. In murine models, we discovered thatHmga1 overexpression is sufficient for clonal expansion and progression to aggressive acute lymphoid leukemia (Cancer Res 2008,68:10121, 2018,78:1890; Nature Comm 2017,8:15008). Further, HMGA1 is overexpressed in pediatric B-ALL (pB-ALL) blasts with highest levels in children who relapse early compared to those who achieve chronic remissions. Together, these findings suggest that HMGA1 is required for leukemogenesis and may foster relapse in B-ALL. We therefore sought to: 1) test the hypothesis that HMGA1 is a key epigenetic regulator required for leukemogenesis and relapse in pB-ALL, and, 2) elucidate targetable mechanisms mediated by HMGA1 in leukemogenesis. Methods: We silenced HMGA1 via lentiviral delivery of short hairpin RNAs targeting 2 different sequences in cell lines derived from relapsed pB-ALL (REH, 697). REH cells harbor the TEL-AML1 fusion; 697 cells express BCL2, BCL3, and cMYC. Next, we assessed leukemogenic phenotypes in vitro (proliferation, cell cycle progression, apoptosis, and clonogenicity) and leukemogenesis invivo. To dissect molecular mechanisms underlying HMGA1, we performed RNA-Seq and applied in silico pathway analysis. Results: There is abundant HMGA1 mRNA and protein in both pB-ALL cell lines and HMGA1 was effectively silenced by short hairpin RNA. Further, silencing HMGA1 dramatically halts proliferation in both cell lines, leading to a decrease in cells in S phase with a concurrent increase in G0/S1. Apoptosis also increased by 5-10% after HMGA1 silencing based on flow cytometry for Annexin V. In colony forming assays, silencing HMGA1 impaired clonogenicity in both pB-ALL cell lines. To assess HMGA1 function in leukemogenesis in vivo, we implanted control pB-ALL cells (transduced with control lentivirus) or those with HMGA1 silencing via tail vein injection into immunosuppressed mice (NOD/SCID/IL2 receptor γ). All mice receiving control REH cells succumbed to leukemia with a median survival of only 29 days. At the time of death, mice had marked splenomegaly along with leukemic cells circulating in the peripheral blood and infiltrating both the spleen and bone marrow. In contrast, mice injected with REH cells with HMGA1 silencing survived for >40 days (P<0.001) and had a significant decrease in tumor burden in the peripheral blood, spleen, and bone marrow. Similar results were obtained with 697 cells, although this model was more fulminant with control mice surviving for a median of only 17 days. To determine whether the leukemic blasts found in mice injected with ALL cells after HMGA1 silencing represented a clone that expanded because it escaped HMGA1 silencing, we assessed HMGA1 levels and found that cells capable of establishing leukemia had high HMGA1 expression, with levels similar to those observed in control cells without HMGA1 silencing. RNA-Seq analyses from REH and 697 cell lines with and without HMGA1 silencing revealed that HMGA1 up-regulates transcriptional networks involved in RAS/MAPK/ERK signaling while repressing the IDH1 metabolic gene, the latter of which functions in DNA and histone methylation. Studies are currently underway to identify effective agents to target HMGA1 pathways. Conclusions: Silencing HMGA1 dramatically disrupts leukemogenic phenotypes in vitro and prevents the development of leukemia in mice. Mechanistically, RNA-Seq analyses revealed that HMGA amplifies transcriptional networks involved cell cycle progression and epigenetic modifications. Our findings highlight the critical role for HMGA1 as a molecular switch required for leukemic transformation in pB-ALL and a rational therapeutic target that may be particularly relevant for relapsed B-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Gain of Function Mutation in the NSD2 Histone Methyltransferase Drives Glucocorticoid Resistance Via Blocking Receptor Auto-Induction and BIM/Bmf Expression in ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3758-3758
Author(s):  
Jianping Li ◽  
Catalina Troche ◽  
Julia Hlavka Zhang ◽  
Jonathan Shrimp ◽  
Jacob S. Roth ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Mutant Allele ◽  
Gene Editing ◽  
Conflicts Of Interest ◽  
Chemotherapeutic Agents ◽  
Rna Seq ◽  
Mesenchymal Transition ◽  
Pediatric All

Despite improvements in chemotherapy that have increased the 5-year survival rates of pediatric ALL to close to 90%, 15-20% of patients may relapse with a very poor prognosis. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biology of mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in B-ALL cell lines (RCH-ACV and SEM) and T-ALL cell line (RPMI-8402) or insert the E1099K mutation into the NSD2WT T-ALL cell line (CEM) and B-ALL cell line (697). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099Kcells demonstrate enhanced cell growth, colony formation and migration. NSD2E1099K mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system in xenografts. To understand why this NSD2 mutations are identified prominently in children who relapse early from therapy for ALL, we performed high-throughput screening in our isogenic cell lines with the National Center for Advancing Translation Science (NCATS) Pharmaceutical Collection and other annotated chemical libraries and found that NSD2E1099K cells are resistant to glucocorticoids (GC) but not to other chemotherapeutic agents used to treat ALL such as vincristine, doxorubicin, cyclophosphamide, methotrexate, and 6-mercaptopurine. Accordingly, patient-derived-xenograft ALL cells with NSD2E1099K mutation were resistant to GC treatment. Reversion of NSD2E1099K mutation to NSD2WT restored GC sensitivity to both B- and T-ALL cell lines, which was accompanied by cell cycle arrest in G1 and induced-apoptosis. Furthermore, knock-in of the NSD2E1099K mutation conferred GC resistance to ALL cell lines by triggering cell cycle progression, proliferation and anti-apoptotic processes. Mice with NSD2E1099K xenografts were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2WT cells led to significant tumor reduction and survival benefit. To illustrate these biological phenotypes and understand the molecular mechanism of GC resistance driven by NSD2E1099Kmutation, we investigated the GC-induced transcriptome, GC receptor (GR) binding sites and related epigenetic changes in isogenic ALL cell lines in response to GC treatment. RNA-Seq showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, especially in T-ALL cell lines, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2WT cells. The critical pro-apoptotic regulators BIM and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that, the NSD2E1099K mutation blocked the ability of GR and CTCF to bind most GC response elements (GREs) such as those within BIM and BMF. While GR binding in NSD2WT cells was accompanied by increased H3K27 acetylation and gene expression, this failed to occur in NSD2 mutant cells. Furthermore, we found that GR RNA and protein levels were repressed in ALL cells expressing NSD2E1099K and GC failed to induce GR expression in these cells. Paradoxically, while H3K27me3 levels were generally decreased in NSD2E1099K cells, we saw increased levels of H3K27me3 at the GRE within the GR gene body where GR itself and CTCF normally bind, suggesting a novel role for the polycomb repressive complex 2 and EZH2 inhibitors for this form of GC resistance. In conclusion, these studies demonstrate that NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by interfering with the GR expression and its ability to bind and activate key target genes. Gene editing screens are being performed to understand how to overcome this resistance. Disclosures No relevant conflicts of interest to declare.

TET2 Downregulation Leads to AML Cells Sensitive to Decitabine

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3643-3643
Author(s):  
Qingxiao Chen ◽  
Jingsong He ◽  
Xing Guo ◽  
Jing Chen ◽  
Li Yang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Methylation Status ◽  
Control Cell ◽  
Gene Array ◽  
Expression Level ◽  
Cell Cycle Profile ◽  
Knock Down ◽  
Specific Pcr

Abstract Background: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and have very lethal rate. Chemotherapy is the main method to treat AML, but the complete remission rate is still not very optimal. With the development of genetic and molecular biology technologies, more and more molecular biomarkers are found, some of them are useful for us to evaluate the prognosis and can help us to tailor the treatment plan for different patients. TET2, a member of the ten-eleven-translocation(TET) family genes which can modify DNA by catalyzing the conversion of 5mehtyl-cytosine(5-mC) to 5-hydroxymethyl-cytosine(5-hmC), is often inactivated through loss-of-function mutation and deletion in myeloid malignancies. Recent clinical research reported that the lower the expression of TET2 in MDS and AML patients, the better the response to decitabine (DAC, a demethylation agent) will be. However, the mechanism of the phenomenon is still unknown. Our investigation is trying to uncover the mechanism how TET2 protein levels are negatively related with AML sensitivity to decitabine. Methods: We detected TET2 mRNA expression level in acute leukemia cell lines, bone marrow AML specimens and peripheral blood mononuclear cells from healthy donors by semiquantitative real time polymerase chain reaction (qRT-PCR). Western blot is also applied to detect TET2 protein expression. In order to access TET2 methylation status, we used the methylation-specific PCR. And we also checked the mutant status of TET2 in U937 and KG-1 cell line. CCK8 and flow cytometry are used to detect cell proliferation rate, cell apoptosis, and cell cycle profile. Also, we developed TET2 knock-down and overexpression lentivirus to transfect AML cell lines to explore the mechanism why TET2 expression level is related to the response of DAC. Last, gene array is used to compare gene expression level changes between TET2 knock-down cell lines (or TET2 overexpression cell lines) and the control cell lines. Results: The AML cell lines (KG-1, U937, Kasumi, HL-60, THP-1) and AML patients specimens express lower TET2 than that of PBMC from the healthy donor (P<0.05). Among AML cell lines, U937 barely expresses TET2, while KG-1 expresses TET2 relatively higher than other AML cell lines. The methylation-specific PCR showed that TET2 in U937 was partially methylated while KG-1 was not. After using decitabine to treat U937 cell line, the TET2 methylation status was attenuated. And all the exons of TET2 were not detected any mutation in KG-1 AND U937. Then, we used CCK8 to compare the response difference to DAC between U937 and KG-1 and found that U937 is much more sensitive to DAC rather than KG-1 (P<0.05). Next, we constructed a TET2 shRNA to transfect KG-1, both qRT-PCR and WB were used to verification the knock-down efficiency. Again, CCK8 told us that KG-1 TET2 knock-down cells was more sensitive to DAC than KG-1 NC cells. Flow cytometry identified that cell cycle profile were altered between KG-1 TET2 knock-down cells and KG-1 NC cells. Gene array (KG-1 TET2 KD and KG-1 NC) showed that the expression levels of cell cycle related genes (e.g. CCNB2,RBL1), DNA replication related genes (e.g. PRIM1, RCF3, FEN1) and many other function genes were changed between the knock-down and control cell line. Conclusion: Our study showed that the sensitivity to decitabine of AML cell lines is related to TET2 expression level, knock-down TET2 in KG-1 can increase its vulnerability to decitbine. And the mechanism may be related to the changing expression levels of the genes which regulating cell cycles and DNA replication. Disclosures No relevant conflicts of interest to declare.

Eukaryotic elongation factor 1 alpha 2 alone is not sufficient to cause tumorigenicity in immortalized and neoplastic ovarian cell lines

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 15051-15051
Author(s):  
A. L. Leiser ◽  
N. Rosales ◽  
D. Spriggs
Keyword(s):  
Cell Cycle ◽  
Epithelial Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Control Cell ◽  
Stable Expression ◽  
Size Difference ◽  
Ovarian Carcinomas ◽  
Anchorage Independent Growth ◽  
And Control

15051 Background: EEF1a2 is amplified and overexpressed in 25% of ovarian tumors. Previous studies have shown EEF1a2 to have oncogenic and tumorigenic properties in rodent fibroblasts. However, its role in the tumorigenesis and behavior of human ovarian carcinomas is not known. Methods: Stable expression of EEF1a2 cDNA in both T80 immortalized human ovarian surface epithelial cells and SKOV-3 cell line was followed by standard proliferation assays, soft agar assays for anchorage independent growth, cell cycle analysis, mouse xenograft injections, evaluation for changes in factors such as VEGF and ras and susceptibility to CDDP. Transient transfections with 3 different siRNA’s to EEF1a2 were performed in both cell lines. Results: Stable expression of EEF1a2 was detected by Western Blot and IP. Compared to vector only control, transfection with pCMVTag2B EEF1a2 vector did not change proliferation in either cell line. Anchorage independent growth was slightly higher in the T80 transfected cells. Cell cycle and expression of VEGF and ras were not different than control. IC50 of CDDP was similar between the transfected and control cell lines. No size difference was seen in SKOV transfected and control xenografts. T80 controls grew larger and more frequent tumors than transfected controls. Transient transfection of T80 with siRNA did not result in changes in anchorage independent growth. Conclusion: EEF1a2 alone is not sufficient to act induce tumorigenicity, affect tumor growth rate or drug susceptibility in human ovarian epithelial cells. Experiments utilizing stable siRNA vectors specific to EEF1a2 are underway, as well the combination of EEF1a2 with recognized ovarian tumor oncogenes. No significant financial relationships to disclose.

Connecting gene expression subtypes of colorectal cancer (CRC) with cell lines and drug resistance.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e14544-e14544
Author(s):  
Eva Budinska ◽  
Jenny Wilding ◽  
Vlad Calin Popovici ◽  
Edoardo Missiaglia ◽  
Arnaud Roth ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Clinical Significance ◽  
Cell Lines ◽  
Drug Response ◽  
Drug Sensitivity ◽  
Expression Profiles ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis

e14544 Background: We identified CRC gene expression subtypes (ASCO 2012, #3511), which associate with established parameters of outcome as well as relevant biological motifs. We now substantiate their biological and potentially clinical significance by linking them with cell line data and drug sensitivity, primarily attempting to identify models for the poor prognosis subtypes Mesenchymal and CIMP-H like (characterized by EMT/stroma and immune-associated gene modules, respectively). Methods: We analyzed gene expression profiles of 35 publicly available cell lines with sensitivity data for 82 drug compounds, and our 94 cell lines with data on sensitivity for 7 compounds and colony morphology. As in vitro, stromal and immune-associated genes loose their relevance, we trained a new classifier based on genes expressed in both systems, which identifies the subtypes in both tissue and cell cultures. Cell line subtypes were validated by comparing their enrichment for molecular markers with that of our CRC subtypes. Drug sensitivity was assessed by linking original subtypes with 92 drug response signatures (MsigDB) via gene set enrichment analysis, and by screening drug sensitivity of cell line panels against our subtypes (Kruskal-Wallis test). Results: Of the cell lines 70% could be assigned to a subtype with a probability as high as 0.95. The cell line subtypes were significantly associated with their KRAS, BRAF and MSI status and corresponded to our CRC subtypes. Interestingly, the cell lines which in matrigel created a network of undifferentiated cells were assigned to the Mesenchymal subtype. Drug response studies revealed potential sensitivity of subtypes to multiple compounds, in addition to what could be predicted based on their mutational profile (e.g. sensitivity of the CIMP-H subtype to Dasatinib, p<0.01). Conclusions: Our data support the biological and potentially clinical significance of the CRC subtypes in their association with cell line models, including results of drug sensitivity analysis. Our subtypes might not only have prognostic value but might also be predictive for response to drugs. Subtyping cell lines further substantiates their significance as relevant model for functional studies.

MLL-Fusion Proteins Induce a Stem Cell Program in Committed Progenitors To Generate Leukemia Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 465-465
Author(s):  
Andrei V. Krivstov ◽  
David Twomey ◽  
Zhaohui Feng ◽  
Matthew C. Stubbs ◽  
Todd R. Golub ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Cell Lines ◽  
Fusion Proteins ◽  
Expression Profiles ◽  
Cellular Reprogramming ◽  
Gene Set Enrichment Analysis ◽  
Myelogenous Leukemia ◽  
Self Renewal ◽  
Leukemia Stem Cell

Abstract Leukemias are composed of a hierarchy of cells only a fraction of which have stem cell like properties, and are capable of self-renewal. MLL fusion proteins produced by translocations involving the Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 confer stem cell-like properties on committed hematopoietic progenitors. This provides an opportunity to determine if global cellular reprogramming is necessary for leukemia stem cell (LSC) generation from committed progenitors or if induction of a more limited self-renewal signature in committed progenitors is sufficient. We transduced murine IL-7R− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIhi granulocyte macrophage progenitors (GMPs) with retroviruses encoding the MLL-AF9 fusion protein, which led to the development of acute myelogenous leukemia. From the leukemias we isolated a population of IL-7R− Lin− Sca-1− c-Kit+ CD34int. FcγRII/IIIint. LSCs which can transplant the disease when fewer than 20 cells are injected into secondary recipients. We used hierarchical clustering, K-means clustering and principal component analysis to compare gene expression profiles of the LSC population to the normal lin− sca-1+ c-kit+ HSC-enriched population, IL-7R− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIlo common myeloid progenitors (CMPs), IL-7R− Lin− Sca-1− c-Kit+ CD34− FcγRII/III− megakaryocyte erythroid progenitors (MEPs) and GMPs and found that the global gene expression profile most resembles the normal GMP from which they arose. However, a leukemia self-renewal signature was identified that shows significant overlap with a group of genes normally highly expressed in HSCs whose expression decreases during the transition to normal committed progenitors. Supervised analysis and gene set enrichment analysis (GSEA) demonstrated approximately 300 genes in the leukemia self-renewal signature. This is only a subset of the approximately 1500 genes that are highly expressed in the normal HSC-enriched population that show decreased expression in CMPs, MEPs, and GMPs. Next, we determined if this 300-gene leukemia stem cell signature is directly regulated by MLL-AF9 or if there is a hierarchy of gene expression. Assessment of gene expression changes 48 hours after MLL-AF9 expression in isolated GMPs demonstrated increased expression of 23/300 genes in the leukemia self-renewal signature. Of interest, there is a high degree of similarity between the 23 MLL immediate response genes and human MLL-rearranged AMLs including HOXA5, HOXA7, HOXA9, HOXA10, MEIS1 and genes not previously known to have a role in MLL-mediated leukemogenesis such as myocyte enhancer factor 2C (MEF2C). Detailed loss-of-function studies using shRNA and dominant negative mutants show inhibition of MEF2C reduces LSC colony formation and serial replating in semi-solid culture to less than 20% of control. Furthermore shRNA mediated inhibition of MEF2C has a significant impact on proliferation of human MLL-AF9 dependent leukemia cell lines, but not cell lines from other subtypes of AML. These data demonstrate LSCs can be generated from committed progenitors without widespread reprogramming of gene expression, and a leukemia self-renewal signature is activated in the process. We have used this program to identify MEF2C as playing a role in MLL-AF9 induced AML. Identification of this program provides an opportunity to further assess its importance in normal tissue homeostasis and neoplastic self-renewal/proliferation, and defines the progression from normal hematopoietic progenitor to leukemia stem cell.

EBV Microrna Mir-BHRF1-2 Targets PRDM1/Blimp1: Potential Role in EBV Lymphomagenesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3547-3547
Author(s):  
Jiao Ma ◽  
Kui Nie ◽  
David Redmond ◽  
Yifang Liu ◽  
Daniel M Knowles ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Binding Site ◽  
Cell Line ◽  
Cell Lines ◽  
Target Genes ◽  
Rna Seq ◽  
B Cell Lymphomas ◽  
Down Regulation ◽  
Over Expression

Abstract PRDM1/Blimp1, a master regulator of B-cell terminal differentiation, has been identified as a tumor suppressor gene in the pathogenesis of diffuse large B-cell lymphoma (DLBCL). In DLBCL, PRDM1 is inactivated by mutations and deletions; however, there is also evidence that PRDM1 is down-regulated by microRNAs (miRNAs) in DLBCL and Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma (cHL). A decrease in PRDM1 activity contributes to the pathogenesis of DLBCL and cHL by inhibiting plasma cell differentiation triggered by signal transduction pathways such as the NF-kB pathway. Since malignant EBV-positive B-cell lymphoproliferations are often associated with increased NF-kB activity, it is conceivable that abnormal PRDM1 down-regulation may play a role in their pathogenesis. EBV-positive B-cell lymphomas are postulated to originate from EBV-infected B-cells with latency III growth program of EBV gene expression. Thus, EBV-immmortalized lymphoblastoid cell lines (LCLs), which are of latency III type, serve as a good model to study EBV lymphomagenesis. We observed discordance in PRDM1 mRNA and protein levels in LCLs. By quantitative real-time reverse transcriptase PCR, PRDM1 mRNA levels in LCLs varied from 14.6% to 1259.7% relative to the multiple myeloma cell line U266, which expresses high levels of PRDM1. However, PRDM1 protein was discordantly low in LCLs compared to U266 based on immunohistochemistry and Western blotting assays, consistent with post-transcriptional regulation. EBV encodes 25 viral miRNAs, and we postulate that one of more of them may function to dampen PRDM1 expression. Indeed, a miRNA binding site containing seed match to bases 2-7 of EBV miR-BHRF1-2 was identified in positions 1565 to 1589 of PRDM1 3’ untranslated region. MiR-BHRF1-2 functionally targeted this specific binding site and repressed luciferase reporter activity. Mutation in the seed region of this site relieved the repression in comparison to the wild type control. MiR-BHRF1-2 was highly expressed in LCLs, while it was barely detectable in the EBV-positive Burkitt lymphoma cell line MUTU I, which has latency type I. Importantly, immunoblotting assay demonstrated an up-regulation of PRDM1 protein level in CCL156 and CCL159 LCL cells transfected with miR-BHRF1-2 inhibitor relative to those transfected with miRNA Inhibitor negative control, supporting a role of miR-BHRF1-2 in PRDM1 down-regulation in vivo. To examine the biological consequences of increased PRDM1 expression in LCL cells, PRDM1 was over-expressed in JY25 and CCL159 LCL cell lines. Enforced expression of PRDM1 induced apoptosis in both cell lines. Furthermore, bromodeoxyuridine (Brdu) incorporation study demonstrated that overexpression of PRDM1 reduced the percentage of S phase from 43.4% to 27.6% in CCL159 cells, and 39.5% to 27.9% in JY25 cells, respectively. Whole transcriptome sequencing (RNA-seq) identified a set of potential PRDM1 direct target genes whose expressions decreased in both LCL cell lines upon PRDM1 over-expression. These genes have broad functions including cell proliferation and survival, transcription and translation, mitochondrial functions, and cytoskeleton. Although no significant changes in cell cycle and apoptosis were observed upon transfection of miR-BHRF1-2 inhibitor, RNA-seq analysis of CLL159 cells transfected with miR-BHRF1-2 inhibitor revealed a small subset of repressed genes which overlapped with those identified by PRDM1 over-expression. This finding suggests that the increase in PRDM1 expression upon miR-BHRF1-2 inhibition, albeit small, is capable of repressing a subset of PRDM1 target genes with potential biological effects. In summary, our findings demonstrate that PRDM1 is a target of EBV miR-BHRF1-2. MiR-BHRF1-2 mediated PRDM1 down-regulation may contribute to the pathogenesis of EBV-associated B-cell lymphomas by inhibiting the transcription repression program of PRDM1 and limiting PRDM1-mediated cellular changes detrimental to tumor growth, including cell cycle arrest and apoptosis. Further characterization of the target genes whose expression is up-regulated by miR-BHRF1-2-mediated PRDM1 down-regulation may provide important clues to the pathogenetic function of miR-BHRF1-2 and EBV oncogenesis in general. Disclosures No relevant conflicts of interest to declare.

scholarly journals High Mobility Group A1 Chromatin Regulators: Key Epigenetic Switches and Therapeutic Targets Required for Leukemic Transformation in JAK2 Mutant MPN

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1680-1680
Author(s):  
Liping Li ◽  
Wenyan Lu ◽  
Alison R. Moliterno ◽  
Lingling Xian ◽  
Joseph Kim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
High Mobility ◽  
Transcriptional Networks ◽  
Murine Models ◽  
Rna Seq ◽  
Leukemic Transformation ◽  
Cycle Progression ◽  
Cd34 Cells

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML). However, mechanisms driving progression remain elusive and therapies are ineffective after leukemia develops. The High Mobility Group A1/2 (HMGA1/2) genes encode oncogenic chromatin remodeling proteins which are overexpressed in aggressive solid tumors where they portend adverse outcomes. HMGA1/2 genes are also up-regulated in hematologic malignancies and MPN with disease progression. In murine models, Hmga1/2 overexpression drives clonal expansion and deregulated proliferation while Hmga1 overexpression is sufficient for lymphoid leukemic transformation. We therefore sought to: 1) test the hypothesis that HMGA1/2 proteins are rational therapeutic targets required for leukemic transformation in MPN, 2) elucidate mechanisms mediated by HMGA1/2 during disease progression, and, 3) identify therapeutic approaches to disrupt HMGA function and intercept the transition from chronic disease to aggressive leukemia. Methods: We compared HMGA1/2 in JAK2V617F mutant AML cell lines from MPN patients (DAMI, SET-2), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F/MPLSV). To elucidate HMGA1/2 function, we silenced HMGA1 or HMGA2 via short hairpin RNA in human MPN-AML cells and generated murine models of PV-AML with heterozygous Hmga1 or Hmga2 deficiency. To dissect molecular mechanisms underlying HMGA, we compared RNA-Seq from MPN-AML cell lines after gene silencing. Finally, to identify therapies to target HMGA pathways, we integrated the RNA-Seq data with the Broad Connectivity Map (cMAP). Results: There is a marked up-regulation in HMGA1/2 in CD34+ cells from PV patients after transformation to AML and in leukemic blasts from our PV-AML mouse model. Conversely, silencing HMGA1 or HMGA2 in human MPN-AML cell lines (DAMI, SET-2) dramatically halts proliferation, disrupts clonogenicity, and prevents leukemia development in mice. Further, heterozygous Hmga1 deficiency prolongs survival in the transgenic PV-AML murine model with fulminant leukemia and early mortality, although Hmga2 deficiency has no effect. RNA-Seq analyses from human MPN-AML cell lines revealed that HMGA1 up-regulates transcriptional networks involved in cell cycle progressions (E2F targets, mitotic spindle, G2M checkpoint, MYC targets) while repressing immune pathways (inflammation, interferon gamma) and oxidative phosphorylation. HMGA2 up-regulates similar pathways, but represses TNFalpha signaling. cMAP identified inhibitors of histone deacetylation and cell cycle progression as potential agents to target HMGA1 pathways; DNA synthesis inhibitors were predicted to target HMGA2 pathways. Cytotoxicity assays demonstrate that epigenetic therapy with HDAC inhibitors synergizes with Ruxolitinib in JAK2 mutant MPN cells after transformation to leukemia. Conclusions: HMGA1/2 genes are overexpressed in MPN with highest levels after leukemic transformation. Further, silencing HMGA1/2 disrupts leukemogenic phenotypes in vitro and prevents the development of leukemia in mice. In addition, heterozygous deficiency of Hmga1 prolongs survival in a fulminant MPN-AML model. Mechanistically, RNA-Seq analyses revealed that HMGA amplifies transcriptional networks involved cell cycle progression, which can be targeted with epigenetic therapies. Our findings further underscore the key role for HMGA as an epigenetic switch required for leukemic transformation in MPN and opens the door to novel therapeutic approaches to intercept the transition from chronic indolent disease to aggressive leukemia. Disclosures No relevant conflicts of interest to declare.

Novel Anthraquinone Derivatives as Dual Inhibitors of Topoisomerase 2 and Casein Kinase 2: In Silico Studies, Synthesis and Biological Evaluation on Leukemic Cell Lines

2019 ◽  
Vol 18 (11) ◽  
pp. 1551-1562 ◽  
Author(s):  
Abbas Kabir ◽  
Kalpana Tilekar ◽  
Neha Upadhyay ◽  
C.S. Ramaa
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Biological Evaluation ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Cell Cycle Analysis ◽  
Protein Targets ◽  
Topoisomerase 2 ◽  
Dual Inhibitors

Background: Cancer being a complex disease, single targeting agents remain unsuccessful. This calls for “multiple targeting”, wherein a single drug is so designed that it will modulate the activity of multiple protein targets. Topoisomerase 2 (Top2) helps in removing DNA tangles and super-coiling during cellular replication, Casein Kinase 2 (CK2) is involved in the phosphorylation of a multitude of protein targets. Thus, in the present work, we have tried to develop dual inhibitors of Top2 and CK2. Objective: With this view, in the present work, 2 human proteins, Top2 and CK2 have been targeted to achieve the anti-proliferative effects. Methods: Novel 1-acetylamidoanthraquinone (3a-3y) derivatives were designed, synthesized and their structures were elucidated by analytical and spectral characterization techniques (FTIR, 1H NMR, 13C NMR and Mass Spectroscopy). The synthesized compounds were then subjected to evaluation of cytotoxic potential by the Sulforhodamine B (SRB) protein assay, using HL60 and K562 cell lines. Ten compounds were analyzed for Top2, CK2 enzyme inhibitory potential. Further, top three compounds were subjected to cell cycle analysis. Results: The compounds 3a to 3c, 3e, 3f, 3i to 3p, 3t and 3x showed excellent cytotoxic activity to HL-60 cell line indicating their high anti-proliferative potential in AML. The compounds 3a to 3c, 3e, 3f, 3i to 3p and 3y have shown good to moderate activity on K-562 cell line. Compounds 3e, 3f, 3i, 3x and 3y were found more cytotoxic than standard doxorubicin. In cell cycle analysis, the cells (79-85%) were found to arrest in the G0/G1 phase. Conclusion: We have successfully designed, synthesized, purified and structurally characterized 1- acetylamidoanthraquinone derivatives. Even though our compounds need design optimization to further increase enzyme inhibition, their overall anti-proliferative effects were found to be encouraging.

scholarly journals Genome-Wide Role of HSF1 in Transcriptional Regulation of Desiccation Tolerance in the Anhydrobiotic Cell Line, Pv11

2021 ◽  
Vol 22 (11) ◽  
pp. 5798
Author(s):  
Shoko Tokumoto ◽  
Yugo Miyata ◽  
Ruslan Deviatiiarov ◽  
Takahiro G. Yamada ◽  
Yusuke Hiki ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Desiccation Tolerance ◽  
Expression Profiles ◽  
Insect Cell Line ◽  
Gene Expression Profiles ◽  
Late Embryogenesis Abundant ◽  
Heat Shock Factor 1 ◽  
Genome Wide ◽  
A Genome

The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.

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