scholarly journals Marcks Is a Critical Downstream Mediator of IL-1-Driven AML Progression

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2679-2679
Author(s):  
Mona M.Hosseini ◽  
Hsin-Yun Lin ◽  
Gabby Dewson ◽  
Ruthey Vivier ◽  
Anupriya Agarwal
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Crucial Role ◽  
Cell Cycle Progression ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Rna Seq ◽  
Cycle Progression ◽  
Genetic Subtypes ◽  
Human Leukemia Cells

Background: Whole-genome sequencing and expression studies have revealed significant heterogeneity in the molecular abnormalities driving AML. Selective inhibitors have been developed for many of the pathways influenced by these genetic alterations, but successful translation of these agents into the clinic is limited by both disease heterogeneity and drug resistance. Targeting of inflammatory pathways to block leukemia progression and eliminate leukemic clones is an emerging concept in AML therapy. We have shown that elevated levels of pro-inflammatory cytokine, interleukin-1 (IL-1), in AML microenvironment enhances the growth of leukemic progenitors in variety of genetic subtypes while inhibiting the normal progenitors' growth. To reveal molecular mechanisms underlying such paradoxical effect, we performed RNA-seq analysis on AML and healthy progenitors post IL-1 stimulation. We found myristoylated alanine-rich C-kinase substrate (MARCKS) is one of the most differentially expressed genes in AML progenitors compared to healthy progenitors. MARCKS is a major substrate of protein kinase C, and plays a crucial role in cell survival, migration, and cell cycle progression. Increased MARCKS expression promotes metastasis in solid tumors and inhibiting its activation is being proposed as a therapeutic strategy. However, its role in AML has not yet been investigated. Here, we show a crucial role of MARCKS activation in IL-1-mediated leukemia progression. Method and Results: Using the RNA-seq gene expression data of 451 primary AML patient samples (Tyner et al., Nature 2018), we tested the correlation of MARCKS with IL1R1 receptor expression in AML primary samples and found it to be positively correlated (r = 0.45, p < 0.0001). The correlation was regardless of sex, age, and mutation status. Using q-PCR and western blot analysis, we showed that MARCKS expression, protein level, and its activation (phosphorylation) are elevated in AML samples at basal level and after IL-1 stimulation when compared to the healthy progenitors (~3 fold change). These results validated our transcriptome data and suggested an important role for MARCKS in IL-1-mediated AML progression. To identify the functional significance of MARCKS in AML, we used two independent doxycycline inducible shRNAs to knockdown MARCKS in AML cell lines (MOLM-14 and THP-1). Our data show that MARCKS depletion in AML cells reduces the cell viability overtime to 40%, cell growth to 4 fold, and colony formation ability to 2 fold. Mechanistically, the knockdown of MARCKS in AML cells decreased SKP2 and increased p27 protein levels, suggesting MARCKS regulates cell cycle progression in these cells. We xenografted MOLM-14 cells expressing MARCKS shRNA into NSG mice by tail vein injections and induced the knockdown in vivo by feeding mice doxycycline containing chow. The bone marrow and spleen cells were analyzed by flow cytometry for human and mouse cell markers approximately 3 weeks post-treatment. We observed that the knockdown of MARCKS decreased the leukemia burden in xenograft model as observed by ~80% reduction in human leukemia cells in the bone marrow, ~40% reduction in human leukemia cells in spleen, and ~50% reduction in spleen size compared to the controls, suggesting MARCKS has a critical role in leukemia progression. Conclusion: MARCKS is over-expressed and -activated in various AML genetic subtypes. IL-1 stimulation of AML progenitors increases MARCKS phosphorylation. MARCKS promotes AML progression by increasing the cellular growth, survival, and cell cycle progression of leukemic cells. These results suggest that MARCKS may serve as marker for IL-1 mediated inflammatory stress and offers a route for new targeted therapy. Disclosures No relevant conflicts of interest to declare.

2,5-Dimethyl-Celecoxib Inhibits Cell Cycle Progression and Induces Apoptosis in Human Leukemia Cells

2015 ◽  
Vol 355 (2) ◽  
pp. 308-328 ◽  
Author(s):  
Cyril Sobolewski ◽  
Jiyun Rhim ◽  
Noémie Legrand ◽  
Florian Muller ◽  
Claudia Cerella ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Cycle Progression ◽  
Human Leukemia Cells

Myb Family Transcription Factors Contribute to G2/M Cell Cycle Transition in Normal and Malignant Hematopoietic Cells by Direct Regulation of Cyclin B1 Expression.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1115-1115
Author(s):  
Yuji Nakata ◽  
Susan Shetzline ◽  
Chizuko Sakashita ◽  
Anna Kalota ◽  
Stephen I. Rudnick ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Hematopoietic Cells ◽  
Cyclin B1 ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
M Cell ◽  
Human Leukemia Cells ◽  
M Phase ◽  
Cell Cycle Transition

Abstract Myb family transcription factors are ubiquitously expressed, and are known to play a critical role in regulating G1/S cell cycle transition. Recently, Myb-like proteins have been found to regulate G2/M transit in plants, yeast, and Drosophila. A recent study in human T98G ganglioblastoma cells revealed that E2F, together with B-Myb, but not c-Myb, regulated cyclin B1 expression directly. However, in hematopoietic cells Myb’s role in regulating cell cycle check points other than G1/S is less well defined. Herein we report that c-Myb, as well as B-Myb, up-regulates cyclin B1 expression in normal and malignant human hematopoietic cells, thereby contributing to G2/M cell cycle progression. Our initial experiments revealed a direct relationship between Myb and cyclin B1 expression. We then attempted to show causality using a variety of experimental approaches. First, ChIP assays demonstrated that c-Myb protein directly bound the cyclin B1 promoter in K562 and Mo7e cells. Second, a cycle 3 GFP reporter construct, driven by the cyclin B1 promoter, was upregulated in cells co-tranfected with a c-myb expression vector. Third, a conditionally active c-Myb restored cyclin B1 mRNA expression in K562 human leukemia cells in presence of cycloheximide in 6 hours. All these assays strongly suggest that c-myb directly regulates cyclin B1. Finally, cyclin B1 expression decreased by 85–90 % in Mo7e human leukemia cells in which c-myb had been silenced with siRNA. siRNA targeted to B-myb also decreased cyclin B1 expression, while neither siRNA species decreased cdc2 or cyclin A. The biologic significance of this relationship was revealed by two independent lines of experimentation. First, silencing B-myb resulted in a delay of cell cycle progression from S to G2/M, and an accumulation of cells in M phase, in HCT116 cells and K562 cells respectively. These abnormalities could be rescued, at least partially, by expression of exogenous c-myb. This observation conflicts with the report that c-Myb does not regulate cyclin B1 or G2/M progression in T98G cells suggesting that Myb functions could well be cell type specific. Additional analysis using PCR array showed that the absence of B-myb decreased the expression of 19 of 84 cell cycle related genes. Exogenous c-myb expression partially rescued 11 genes including cyclin B1, cyclin B2, cdc2, cdc20, CKS1B, p15INK4b and Ki-67, but not cyclin D1. In another experiment an inducible dominant negative c-Myb protein decreased cyclin B1 expression in K562 human leukemia cells, and the expected consequence of this, accelerated exit from the M phase, was observed. In activated primary human T-lymphocytes with IL-2 and CD34+ bone marrow cells, expression of c-Myb and cyclin B1 increased concordantly and silencing c-myb expression resulted in decreased cyclin B1 expression. We conclude from these studies that c-Myb in addition to B-myb plays a heretofore unappreciated role in G2/M cell cycle transition in normal and malignant human hematopoietic cells by directly regulating cyclin B1 expression.

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 Pterostilbene induces cell cycle arrest and apoptosis in MOLT4 human leukemia cells

2012 ◽  
Vol 50 (4) ◽  
pp. 574-580 ◽  
Author(s):  
Kamila Siedlecka-Kroplewska ◽  
Agnieszka Jozwik ◽  
Lucyna Kaszubowska ◽  
Anna Kowalczyk ◽  
Wojciech Boguslawski
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Cycle Arrest ◽  
Human Leukemia Cells
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