scholarly journals Synergistic Effect of DHODH Inhibitor PTC299 with DNA Demethylating Agent Decitabine in Myelodysplastic Syndromes

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5407-5407
Author(s):  
Kensuke Kayamori ◽  
Cheng Zhong ◽  
Daisuke Shinoda ◽  
Shuhei Koide ◽  
Motohiko Oshima ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Cell Lines ◽  
De Novo ◽  
Enrichment Analysis ◽  
S Phase ◽  
Rna Seq ◽  
Expression Levels ◽  
Pyrimidine Synthesis ◽  
De Novo Pyrimidine Synthesis

Background:Myelodysplastic Syndrome (MDS) is a clonal bone marrow disorder characterized by ineffective and clonal hematopoiesis accompanied by morphological dysplasia and variable cytopenia. There are few treatment options for MDS, and allogenic hematopoietic stem cell transplantation is the only curative option. Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine synthesis, the conversion of DHO to orotate. DHODH inhibition has been described recently as a new approach for treating acute myeloid leukemia (AML) by inducing differentiation of diverse AML subclasses. PTC299 represents a novel potent DHODH inhibitor and recently clinical development of PTC299 as a potential treatment option for acute leukemia was initiated. Here, we explored the efficacy of DHODH inhibitor PTC299 for MDS. Methods:Anti-MDS efficacy of PTC299 was studied using human MDS cell lines and primary MDS cells in vitro. PTC299 was synthesized at PTC Therapeutics Inc. Mechanistic studies were conducted via flow cytometric analysis and RNA-sequencing (RNA-seq). Gene expression levels were analyzed by quantitative PCR (qPCR). Results:PTC299 inhibited proliferation of AML cell lines and induced their differentiation. As previously reported in other DHODH inhibitors, upregulation of CD11b was observed after PTC299 treatment in both HL-60 and THP-1 cells. In addition, PTC299 inhibited the proliferation of MDS cell lines, MDSL and SKM-1 cells, with EC50s of 12.6 nM in MDSL cells and 19.7 nM in SKM-1 cells. The inhibitory effect was reversed by the exogenous addition of 100 µM uridine, which bypasses the requirement for de novo pyrimidine synthesis by feeding into the salvage pathway, thereby negating the need for DHODH. Because the basal expression levels of CD11b are high in MDS cells, we examined the expression levels of CD38. Both cell lines showed dose-dependent upregulation of CD38 after PTC299 treatment. To investigate the possible synergism between PTC299 and decitabine, we treated MDSL and SKM-1 cells with increasing concentrations of PTC299 and decitabine as single agents or in combination. After 3 days of culture, cells were analyzed by MTS assays. PTC299 and decitabine exerted a enhanced cytotoxic effect on MDSL and SKM-1 cells. Similar results were obtained with primary MDS samples. In Annexin/PI assays, the percentage of apoptotic cells was increased by combination of PTC299 with decitabine in both cell lines. Mechanistically, treatment with PTC299 induced an intra-S-phase arrestfollowed by entry intoapoptotic cell death. It has also been reported that the expression of p53 is increased in response to the intra-S-phase arrest. To understand the genome-wide effects and target genes of PTC299 and the combination with decitabine, we performed RNA-seq of MDSL and SKM-1 cells treated with PTC299, decitabine, or the combination of both agents versus DMSO-treated cells. Gene set enrichment analysis (GSEA) using our RNA-seq data confirmed that MYC target gene sets were negatively enriched in both PTC299-, decitabine- and combination- treated cells. KEGG pathway enrichment analysis revealed activation of genes associated with apoptosis in both cell lines. To better elucidate a synergistic effect of PTC299 and decitabine, we performed qPCR of CDKN1A, which is a major target of p53 activity. The mRNA expression levels of CDKN1A were upregulated after treatment with PTC299, which was further enhanced by the combination with decitabine. Conclusions:Our result indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells in vitro and acts in at least an additive and possibly synergistic manner with decitabine in this process. This combination therapy could be a new therapeutic option for the treatment of MDS. Disclosures Lennox: PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment. Sheedy:PTC Therapeutics: Employment.

Leflunomide Inhibits Proliferation of Neoplastic B-Cell Lines and Induces Apoptosis in Primary CLL Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2497-2497
Author(s):  
Andrew P. Mone ◽  
John C. Byrd
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Untreated Control ◽  
Cytotoxic Effect ◽  
De Novo ◽  
Antiproliferative Effect ◽  
Pyrimidine Synthesis ◽  
De Novo Pyrimidine Synthesis

Abstract Leflunomide is a commercially available immunosuppressive agent approved for the treatment of rheumatoid arthritis. The compound is administered orally and is rapidly converted to the active compound A77 1726. The half-life of A77 1726 is long, with values reported as 10–15 days. Steady state plasma concentrations, in the treatment of rheumatoid arthritis, approach 250 μM. The proposed mechanism of A77 1726 is a reduction in lymphocyte proliferation due to inhibition of de novo pyrimidine synthesis and inhibition of tyrosine kinases. Given the need for chemotherapeutics with activity against neoplastic B-cell diseases that possess favorable pharmacokinetic and side effect profiles, we examined the in vitro antiproliferative effect of A77 1726 upon neoplastic B-cell lines, and its in vitro cytotoxic effect upon primary CLL cells. Raji, Ramos, 697, WaC3CD5 and Daudi B-cell lines were treated with A77 1726 (0, 1, 10, 50, 100, 200 and 300 μM) for 24, 48 and 96 hrs in RPMI media supplemented with 10% fetal bovine serum. The antiproliferative effect of was determined using the MTT assay. A77 1726 IC50 values for each cell line are: Raji (36 μM), Ramos (18 μM), 697 (29 μM), WaC3CD5 (83 μM) and Daudi (13 μM). Cell cycle analysis of Raji cells using propidium iodide (PI) staining with FACS analysis, showed a reduction of the fraction of cells in G2 from 19 % to 4.9 % with A77 1726 (200 μM) treatment. A77 1726 binds to albumin, diminishing its effective concentration. Human albumin (3 gm/dl) reduced the effectiveness of A77 1726 (200 μM) upon both Raji and WaC3CD5 cell lines. In the presence of albumin, the number of viable Raji cells increased from 32% to 74%, and for WaC3CD5 cells the value increased from 19% to 78%, as compared to the untreated control cells. Both cell lines multiplied slower in the presence of human albumin, thus the observed antiproliferative effect of A77 1726 was proportionally reduced. A77 1726 reduces de novo pyrimidine synthesis by inhibiting the enzyme dihydroorotate dehydrogenase. The reduction in de novo pyrimidine synthesis can be surmounted by the exogenous addition of uridine to the culture media. The Raji and WaC3CD5 cell lines were incubated with A77 1726 with and without uridine to determine the role of pyrimidine synthesis in A77 1726′s antiproliferative effect upon the cell lines. For the Raji cell line, addition of 50 μM uridine to A77 1726 (200 μM) treated cells increased the number of viable cells from 22% to 62% of the untreated control. For the WaC3CD5 cell line, the addition of uridine did not decrease the antiproliferative effect of A77 1726. These data agree with prior studies that indicated an antiproliferative effect of A77 1726 beyond its inhibition of de novo pyrimidine synthesis. CLL cells do not reproduce in vitro; however, we hypothesized that the pyrimidine-independent effect of A77 1726 may be cytotoxic to CLL cells in vitro. Five negatively selected primary CLL samples were treated with A77 1726 (0, 50, 100, 200 and 300 μM) for 120 hrs and cell viability was determined with the MTT assay. Significant (greater than 40 % of control value) cytotoxicity occurred with 200 μM A77 1726 in 3 of 5 samples. Treatment with 300 μM A77 1726 led to significant killing in all the samples; the mean viability, as compared to untreated control, was 36% (SD 21, N=5). Addition of uridine did not reverse the observed cytotoxic effect of A77 1726 upon CLL cells. Annexin V-FITC/PI staining with FACS analysis confirmed the cytotoxic effect of A77 1726 on CLL cells. Further study of leflunomide in animal models of neoplastic B-cell disorders is warranted.

scholarly journals Function and Evolution of Plasmid-Borne Genes for Pyrimidine Biosynthesis in Borrelia spp

2006 ◽  
Vol 188 (3) ◽  
pp. 909-918 ◽  
Author(s):  
Jianmin Zhong ◽  
Stephane Skouloubris ◽  
Qiyuan Dai ◽  
Hannu Myllykallio ◽  
Alan G. Barbour
Keyword(s):  
Thymidylate Synthase ◽  
De Novo ◽  
Linear Plasmid ◽  
Borrelia Hermsii ◽  
Borrelia Garinii ◽  
E Coli ◽  
Pyrimidine Synthesis ◽  
De Novo Pyrimidine Synthesis

ABSTRACT The thyX gene for thymidylate synthase of the Lyme borreliosis (LB) agent Borrelia burgdorferi is located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agent Borrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of the B. hermsii and B. burgdorferi thyX gene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficient Escherichia coli mutant, and in vitro, by testing their activities after purification. The B. hermsii thyX gene complemented the thyA mutation in E. coli, and purified B. hermsii ThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, the B. burgdorferi ThyX protein had only weakly detectable activity in vitro, and the B. burgdorferi thyX gene did not provide complementation in vivo. The lack of activity of B. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. The B. hermsii thyX locus was further distinguished by the downstream presence in the plasmid of orthologues of nrdI, nrdE, and nrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agents B. burgdorferi and Borrelia garinii. Phylogenetic analysis suggested that the nrdIEF cluster of B. hermsii was acquired by horizontal gene transfer. These findings indicate that Borrelia spp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.

Correlation of radiosensitivity of lung carcinoma to CD137L endogenous expression levels.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14229-e14229
Author(s):  
Swati Girdhani ◽  
Andrew Rodriguez ◽  
Rajiv Dua ◽  
Renate Parry
Keyword(s):  
Cell Proliferation ◽  
Radiation Therapy ◽  
Synergistic Effect ◽  
Tumor Cell ◽  
Cell Lines ◽  
Background Radiation ◽  
Nsclc Cell ◽  
Expression Levels ◽  
Endogenous Expression

e14229 Background: Radiation therapy is a cornerstone in the management of cancer. Preclinical and clinical evidence indicate the therapeutic efficacy of radiation therapy can be augmented by immunomodulatory antibodies against targets such as CD137 and PD-1/PD-L1. Despite evidence of synergy, the mechanistic basis has yet to be fully explored. In this study, we investigated how the expression of CD137, CD137L, PD-1, and PD-L1 correlates with radiosensitivity and proliferation of lung tumor cell lines treated with radiation as well as anti-PD-L1 and agonist anti-CD137 antibodies. Methods: Expression of CD137, CD137L and PD-L1 was measured by RT-qPCR in eight NSCLC cell lines (HCC827, HCC2935, H358, H460, A549, H596, H1650, H520) treated with radiation in doses of 0-8 Gy. Clonogenic assays for radiosensitivity and the CyQUANT assay for cell proliferation were performed after treatment with radiation in combination with anti-PD-L1 (0.01-10µg/ml) and agonist anti-CD137 (0.01-10µg/ml) antibodies. Results: Out of the eight cell lines tested, four were found to be radioresistant – H460, A549, HCC827 and H358 had SF2 (survival at 2 Gy) = 0.76, 0.70, 0.67 and 0.62, respectively – two were moderately sensitive – H520 and HCC2935 (SF2 = 0.54 and 0.40) and two were sensitive to radiation – H596 and H1650 (SF2 = 0.38 and 0.27). Expression analysis by RT-qPCR showed high endogenous expression of CD137L in three out of four radioresistant cell lines while the fourth, H358, had low endogenous expression of CD137L but upon irradiation showed more than a 3-fold induction of CD137L expression. PD-L1 expression levels measured by RT-qPCR did not correlate with radiosensitivity. In vitro cell proliferation was found to be inhibited by 48-68% in all eight NSCLC cell lines after 4 Gy radiation, but combination with anti-PD-L1 and agonist anti-CD-137 antibodies did not contribute additional toxicity. Conclusions: Our results demonstrate that CD137L expression on NSCLC cell lines correlates with radioresistance. Further, addition of anti-PD-L1 and agonist anti-CD137 antibodies to radiation showed little synergistic effect in vitro on tumor cell proliferation. Additional studies to address the synergistic effect of radiation and immunomodulation on tumor cells co-cultured with immune cells is ongoing.

scholarly journals Feedback Responses of the Pyrimidine Metabolism Network Mediate Resistance to Decitabine and 5-Azacytidine

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 537-537
Author(s):  
Xiaorong Gu ◽  
Rita Tohme ◽  
Benjamin K. Tomlinson ◽  
Lisa Durkin ◽  
Caroline Schuerger ◽  
...  
Keyword(s):  
Median Survival ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Metabolic Adaptation ◽  
Pyrimidine Metabolism ◽  
Synthesis Inhibitor ◽  
Pyrimidine Synthesis ◽  
Metabolism Enzymes ◽  
De Novo Pyrimidine Synthesis

There is a need to understand and counter mechanisms-of-resistance to decitabine (Dec) and 5-azacytidine (5Aza), the only agents approved to treat all subtypes of myelodysplastic syndromes (MDS). Both Dec and 5Aza are pro-drugs, and here, in vitro, in vivo and patient data show that while processing the pro-drugs, pyrimidine metabolism is itself altered, dampening pro-drug conversion and causing resistance. Anticipation of these adaptive responses of the pyrimidine metabolism network enables their exploitation instead. Dec and 5Aza are processed by pyrimidine metabolism into a nucleotide analog that depletes DNA methyltransferase 1 (DNMT1). We found that DNMT1 protein, although substantially depleted (~50%) in patients' bone marrows at time-of-response, rebounded at time-of-relapse (Fig 1A). The pyrimidine metabolism enzymes DCK and UCK2 rate-limit cellular uptakes of Dec and 5Aza, respectively. At relapse on Dec (n=13), DCK was halved vs patients' baseline bone marrows (Fig 1B). At relapse on 5Aza (n=14), it was UCK2 that was halved (Fig 1B). Simultaneous with DCK or UCK2 suppression, however, the other enzyme (UCK2 or DCK) was upregulated up to 40-fold (Fig 1B). Seesaw DCK and UCK2 expression was observed also in DCK knock-out (KO) and UCK2-KO AML cells (HAP1): DCK-KO cells upregulated UCK2, resisted Dec, but were more sensitive to 5Aza. UCK2-KO cells upregulated DCK, resisted 5Aza, but were more sensitive to Dec (concentrations for 50% growth inhibition [GI50] Dec: HAP1 wildtype 3mM, DCK-KO 12mM, UCK2-KO 0.1mM; 5Aza: wildtype 4mM, DCK-KO 2mM, UCK2-KO 15mM). These compensatory upregulations of UCK2 or DCK were adaptive to DCK- or UCK2-KO-induced decreases in dCTP or dTTP respectively. We thus examined if Dec or 5Aza also perturbed dCTP/dTTP amounts to trigger adaptive metabolic shifts. Dec decreased dTTP and increased dCTP, while 5Aza decreased dCTP, 24 hours after their addition to AML cells (THP1, OCI-AML3, MOLM13, K562)(Fig 1C). A single dose of Dec or 5Aza was also sufficient to upregulate UCK2 or DCK >2-fold, and the catabolic enzyme CDA >4-fold, within 72 hours (Fig 1D). Protein levels tracked the mRNA changes. Exponentially proliferating Dec or 5Aza-resisting AML cells emerged within 35 days in presence of pro-drug (5 AML cell lines) and displayed the same metabolic reconfigurations seen acutely. We then used patient-derived xenotransplant (PDX) models of chemorefractory AML to identify methods to counter this automatic metabolic adaptation or 'auto-resistance'. The Dec and 5Aza doses used (subcutaneous Dec 0.2 mpk or 5Aza 2 mpk) were chosen to deplete DNMT1 from marrow without cytotoxicity, demonstrated by flow cytometry for DNMT1 and gH2AX. We found (i) Dec timed to avoid DCK troughs (D1 & 2 each week) was superior to Dec timed to coincide with DCK troughs (D1 & 4 each week)(median survival 74 vs 62 days [vehicle 40 days], Log-rank p=0.006); (ii) Adding a CDA inhibitor (intraperitoneal tetrahydrouridine 4-10 mpk) to halved doses of Dec or 5Aza (0.1 mpk, 1 mpk respectively, to avoid cytotoxicity) was superior to no CDA inhibitor + Dec 0.2 mpk or 5Aza 2 mpk (median survival 180 vs 115 days [vehicle 50 days], Log-rank p=0.004), and (iii) Alternating Dec and 5Aza timed to peaks of mutual priming (e.g., D1 & 4 each week) was superior to Dec alone or 5Aza alone, or Dec/5Aza alternating every month or Dec/5Aza given together - the regimen incorporating these lessons extended survival by several months vs vehicle (median survival 223 vs 50 days, Log-rank p=0.003). Eventual AML relapse/progression on this therapy was again caused by AML cells in which DNMT1 was not depleted (Fig 1E), because of even larger increases in CDA and upregulation of de novo pyrimidine synthesis. We are thus evaluating higher CDA-inhibitor doses (40 mpk) and incorporation of non-cytotoxic doses of a de novo pyrimidine synthesis inhibitor. In sum, the pyrimidine metabolism network responds automatically to Dec or 5Aza perturbation with adaptive shifts that decrease pro-drug conversion into the DNMT1-depleting nucleotide. These metabolic shifts can be anticipated (Fig 1F) and exploited, using simple, clinically viable treatment modifications. Disclosures Maciejewski: Novartis: Consultancy; Alexion: Consultancy. Saunthararajah:EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties; Novo Nordisk: Consultancy.

scholarly journals L-Asparaginase Causes Metabolic Reprogramming in ALL Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 922-922
Author(s):  
Ivana Hermanova ◽  
Karel Valis ◽  
Hana Nuskova ◽  
Meritxell Alberich-Jorda ◽  
Amaia Arruabarrena Aristorena ◽  
...  
Keyword(s):  
Cell Lines ◽  
De Novo ◽  
Protein Translation ◽  
Metabolic Reprogramming ◽  
Leukemic Cells ◽  
Wild Type ◽  
Cell Respiration ◽  
Pyrimidine Synthesis ◽  
Mtorc1 Pathway ◽  
De Novo Pyrimidine Synthesis

Abstract Acute lymphoblastic leukemia (ALL) is the most frequent type of childhood cancer. The key component in the therapy, L-asparaginase (ASNase), hydrolyzes plasma asparagine and glutamine. Leukemic cells are sensitive to the depletion due to low activity of asparagine synthetase. Although the treatment is very effective, resistance and side effects remain a serious problem in some cases and its mechanism of action is not well understood. Our aim is to clarify the intracellular consequences of the amino acid depletion to define the reason of different patients´ response. We have generated ASNase-resistant subclones through chronic exposure to the enzyme. Pathway analysis of gene expression profiles of the cell lines (REH;TEL/AML1-positive, NALM-6; TEL/PDGFRB1-positive and their resistant counterparts) and primary samples (sensitive and resistant to ASNase; Holleman et al. (NEJM, 2004)) revealed that ASNase affects the translation machinery and metabolism of leukemic cells. The key nutrient sensor positively regulating protein synthesis, pyrimidine synthesis, glycolysis and lipid synthesis is mTORC1. Since ASNase depletes glutamine that is essential for mTORC1 activity, we hypothesized that the effect of ASNase is driven through mTORC1 signaling. Main aim of the study was to explore the effect of ASNase on downstream mTORC1 targets in leukemic cells. ASNase treatment inhibited protein synthesis, displayed by dephosphorylation of p-P70SK6 and p-S6. ASNase also decreased de-novo DNA synthesis as shown by dephosphorylation of p-CAD. This result was confirmed by analysis of de-novo pyrimidine synthesis intermediates, uridine monophosphate and uridine, measured by UPLC-ToF-MS. Both were decreased upon ASNase treatment. Except experiments done on ALL cell lines we also detected dephosphorylation of p-S6, p-CAD in primary samples. Regarding the effect on glycolysis we observed inhibition of glucose uptake and decrease of lactate production in cells treated with ASNase. We also detected the decrease of protein levels of c-Myc, the activator of glucose and glutamine catabolism, and glucose transporter 1 (Glut-1). On the contrary, ASNase increased fatty acid oxidation (FAO) followed by the elevation of the capacity of cell respiration and NAD+/NADH ratio in both cell lines. Next, we wanted to elucidate whether ASNase inhibits mTORC1 targets through RagB, the key protein mediating response to general amino acid deprivation. We established a RagB mutant leukemic cell line with constitutive activation of mTORC1 pathway despite deprivation of amino acids. ASNase treatment did not inhibit p-S6 and p-CAD in this cell line. Similarly, ASNase failed to increase FAO in cells with active mTORC1. These results suggest that the effect of ASNase on protein translation, de novo pyrimidine synthesis and FAO is mediated through RagB-mTORC1 pathway. By contrast, c-Myc expression was decreased in both RagB wild type and mutant cells, indicating that ASNase inhibits glycolysis in RagB-mTORC1 independent manner. The activation of FAO has been suggested to have a pro-survival function in leukemic cells under nutrient stress conditions. We tested whether the increase of FAO in ALL cells treated with ASNase also serves to cope with the metabolic stress. Pharmacological inhibition of FAO significantly increased the sensitivity of ALL cells to ASNase. Moreover, cells with the inability to increase FAO (RagB mutant) were more sensitive to ASNase compared to RagB wild type cells. Our results show that the inhibitory effect of ASNase on mTORC1 leads not only to apoptosis but also to metabolic reprogramming. We propose that inhibition of protein translation and pyrimidine synthesis are part of apoptotic processes whereas increased FAO and cell respiration represent pro-survival pathway. Altogether, our study suggests that targeting of FAO in leukemic cells resistant to ASNase is a promising new therapeutic strategy. IGA-NT1249, GAUK-632513 Disclosures No relevant conflicts of interest to declare.

scholarly journals Cellular pyrimidine imbalance triggers mitochondrial DNA–dependent innate immunity

2021 ◽  
Author(s):  
Hans-Georg Sprenger ◽  
Thomas MacVicar ◽  
Amir Bahat ◽  
Kai Uwe Fiedler ◽  
Steffen Hermans ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Cultured Cells ◽  
De Novo ◽  
Metabolic Response ◽  
Interferon Response ◽  
Type I ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
De Novo Pyrimidine Synthesis

AbstractCytosolic mitochondrial DNA (mtDNA) elicits a type I interferon response, but signals triggering the release of mtDNA from mitochondria remain enigmatic. Here, we show that mtDNA-dependent immune signalling via the cyclic GMP–AMP synthase‒stimulator of interferon genes‒TANK-binding kinase 1 (cGAS–STING–TBK1) pathway is under metabolic control and is induced by cellular pyrimidine deficiency. The mitochondrial protease YME1L preserves pyrimidine pools by supporting de novo nucleotide synthesis and by proteolysis of the pyrimidine nucleotide carrier SLC25A33. Deficiency of YME1L causes inflammation in mouse retinas and in cultured cells. It drives the release of mtDNA and a cGAS–STING–TBK1-dependent inflammatory response, which requires SLC25A33 and is suppressed upon replenishment of cellular pyrimidine pools. Overexpression of SLC25A33 is sufficient to induce immune signalling by mtDNA. Similarly, depletion of cytosolic nucleotides upon inhibition of de novo pyrimidine synthesis triggers mtDNA-dependent immune responses in wild-type cells. Our results thus identify mtDNA release and innate immune signalling as a metabolic response to cellular pyrimidine deficiencies.

Gli2-regulated activation of hepatic stellate cells and liver fibrosis by TGF-β signaling

2021 ◽  
Author(s):  
Junyan Yan ◽  
Baowei Hu ◽  
Wenjie Shi ◽  
Xiaoyi Wang ◽  
Jiayuan Shen ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Conditional Knockout ◽  
Rna Seq ◽  
Expression Levels ◽  
Liver Fibrogenesis ◽  
Hh Signaling ◽  
Signaling Activity

The Hedgehog (Hh) signaling pathway is correlated with hepatic stellate cells (HSCs) activation and liver fibrosis. Gli2 is a key transcription effector of Hh signaling. However, the role of Gli2 in HSC-mediated liver fibrosis progression is largely unknown. In the present study, we investigated the effect of Gli2 on liver fibrogenesis and its possible mechanism using conditional knockout (cKO) Gli2 mice and HSC models. Wild-type (WT) and GFAP-CreERT;Gli2flox/flox male mice were exposed to CCl4 for one month to induce liver fibrosis. Primary HSCs were isolated from mice and the transition of HSCs into a myofibroblastic phenotype was evaluated. Livers from mice underwent histological, immunohistochemical, and immunofluorescence analyses. The expression levels of proteins and genes were evaluated by Western blot (WB) analysis and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. RNA-seq was used to screen differentially expressed genes. Results showed that CCl4 treatment induced liver fibrosis, promoted HSCs activation and proliferation, and up-regulated Hh signaling activity. The cKO of Gli2 in GFAP-CreERT;Gli2flox/flox mice decreased liver fibrosis as well as HSC activation and proliferation. In vitro studies showed that KO of Gli2 in HSCs blocked cell proliferation and activation by decrease of cyclin D1/D2 expression. The RNA-seq results revealed that the expression levels TGF-β1 ligands were down-regulated in Gli2 KO HSCs. Furthermore, overexpression of Gli2 rescued proliferation and activation of HSCs by up-regulation of TGF-β signaling activity. Our data demonstrated that Gli2 regulated HSC activation and liver fibrosis by TGF-β signaling, thus providing support for future Gli2-based investigations of liver fibrosis therapy.

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.

Targeting RNF8 Effectively Reverse Cisplatin and Doxorubicin Resistance in Endometrial Cancer

2020 ◽  
Author(s):  
Ben Yang ◽  
Wang Ke ◽  
Yingchun Wan ◽  
Tao Li
Keyword(s):  
Endometrial Cancer ◽  
Cell Lines ◽  
Quantitative Polymerase Chain Reaction ◽  
Mrna Levels ◽  
Mice Model ◽  
Expression Levels ◽  
Gynecological Malignancy ◽  
Ec Cells

Abstract Background Endometrial cancer (EC) is one of the most frequent gynecological malignancy worldwide. However, resistance to chemotherapy remains one of the major difficulties in the treatment of EC. Thus, there is an urgent requirement to understand mechanisms of chemoresistance and identify novel regimens for patients with EC. Methods Cisplatin and doxorubicin resistant cell lines were acquired by continuous exposing parental EC cells to cisplatin or doxorubicin for 3 months. Cell viability was determined by using MTT assay. Protein Expression levels of protein were examined by western blotting assay. mRNA levels were measured by quantitative polymerase chain reaction (qPCR) assay. Ring finger protein 8 (RNF8) knockout cell lines were generated by clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene editing assay. Nonhomologous end joining (NHEJ) efficiency were quantified by plasmid based NHEJ assay. DNA double strand breaks (DSB) were generated using laser micro-irradiation. Protein recruitment to DSB was analyzed by immunofluorescent assay. Tumor growth was examined by AN3CA xenograft mice model. Results We found that protein and mRNA expression levels of RNF8 were significantly increased in both cisplatin and doxorubicin resistant EC cells. Cell survival assay showed that RNF deficiency significantly enhanced the sensitivity of resistant EC cells to cisplatin and doxorubicin (P < 0.01). In addition, chemoresistant EC cells exhibited increased NHEJ efficiency. Knockout of RNF8 in chemoresistant EC cells significantly reduced NHEJ efficiency and prolonged Ku80 retention on DSB. Moreover, cisplatin resistant AN3CA xenograft showed that RNF8 deficiency overcame cisplatin resistance. Conclusions Our in vitro and in vivo assays provide evidence for RNF8, which is a NHEJ factor, serving as a promising, novel target in EC chemotherapy.

Sign in / Sign up

Export Citation Format

Share Document