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.

Half-lantern Cyclometalated Platinum(II) Complexes as Anticancer Agents: Molecular docking, Apoptosis, Cell Cycle Analysis and Cytotoxic Activity Evaluations

2021 ◽  
Vol 21 ◽  
Author(s):  
Fatemeh Hajipour ◽  
Masoud Mahdavinia ◽  
Masood Fereidoonnezhad
Keyword(s):  
Cell Cycle ◽  
Molecular Docking ◽  
Comet Assay ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Human Lung ◽  
Cancer Cell Line ◽  
Cell Cycle Analysis ◽  
Mcf 7

Background and Objective: In the design of modern metal-based anticancer drugs, platinum-based complexes have gained growing interest. In this study, the anticancer activity of half-lantern cyclometalated Pt(II)‒Pt(II) complexes were was evaluated using MTT, apoptosis, cell cycle analysis, and DNA binding studies. Materials and Methods: The cytotoxicity of Pt(II)‒Pt(II) complexes were evaluated against different cancer cell lines such as human lung (A549), breast (MCF-7, and MDA-MB-231), ovarian (SKOV-3), and colon (HT-29) as well as normal breast (MCF-10A), and human lung fibroblast MRC-5 cells using MTT assay. BioLegend's PE Annexin V Apoptosis Detection Kit with 7AAD was applied to assess the apoptotic effects of 1A, and 1B compound against MCF-7, and A549 cell lines. Cell cycle analysis was determined using the flowcytometry method. The interaction of compounds with four different DNA structures with PDB codes (1BNA, 1LU5, 3CO3, and 198D) has been investigated by molecular docking. To achieve binding to DNA experimentally, the electrophoresis mobility shift assay and comet assay was applied. Results: In the evaluation of cytotoxic effects, 1A showed the highest cytotoxicity among the studied compounds, and it showed higher potency with more selectivity against normal cell lines than cisplatin. This compound had IC50 of 7.24, 2.21, 1.18, 2.71, 10.65, 18.32 and 49.21 μM against A549, SKOV3, HT29, MCF-7, MDA-MB-231, MRC-5, and MCF-10A, respectively, whereas cisplatin had IC50 of 9.75, 19.02, 107.23, 15.20, 18.09, 14.36, and 24.21 μm, respectively, on the same cell lines. In order to check the DNA binding activity of 1A, and 1B, electrophoretic mobility was also conducted, which indicated that the binding of these compounds led to a slight change in electrophoretic mobility to DNA. The migration of chromosomal DNA from the nucleus in the form of a tail or comet was executed in the comet assay of 1A on MCF-7. Examination of apoptosis of 1A, and 1B on the MCF-7 cancer cell line, showed that it could increase induction of apoptosis in this cancerous cell in a concentration-dependent manner. Investigating the effect of 1A using cell cycle analysis on MCF-7 cancer cell line showed that this complex affects the stage G1 and S of the cell cycle. Conclusion: 1A has the potential to play a significant role in future biopharmaceutical studies.

Design, Synthesis and Biological Evaluation of A Boron Containing Retinoid As a Novel Therapeutic Agent for Acute Promyelocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5008-5008
Author(s):  
Carolina D. Schinke ◽  
Bhaskar C Das ◽  
Swati Goel ◽  
Tushar Bhagat ◽  
Sangeeta Nischal ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Cell Lines ◽  
Therapeutic Agent ◽  
Fold Increase ◽  
Resistant Cell ◽  
Biological Evaluation ◽  
Promyelocytic Leukemia

Abstract Abstract 5008 Cure rates of acute leukemia remain poor emphasizing the need to for novel therapies. All trans retinoic acid (ATRA) is an effective therapeutic agent in a subtype of acute myeloid leukemia and relieves transcriptional repression induced by the PML-RAR oncoprotein by binding to the retinoic acid receptor. Even though ATRA is effective, the treatment course is characterized by a high rate of toxicity and ATRA resistance is seen in some cases of acute promyelocytic leukemia. In an attempt to improve outcomes, we devised a methodology for creation of boronic acid and other newer retinoic acid analogues. Our lead compound, MA-21 was generated by replacing the terminal carboxyl group of ATRA with a boronic ester using Wittig reactions. Computation modeling revealed that MA-21 can fit in the RARa pocket and can form increased covalent bonding with cysteine residues within the receptor. As opposed to other synthetic retinoids, the addition of a boron atom resulted in significantly enhanced cytotoxicity in leukemic cell lines, even those that were resistant to ATRA. MA-21 at 1uM dose led to significant reduction in proliferation of ATRA sensitive NB4 APL cell line (1.8 fold decrease after 96hrs, p= 0.028) as well as in ATRA resistant cell lines NB4.007/6 (3.3 fold decrease after 72hrs and 2.1 decrease after 96hrs, p values of 0.018 and 0.046) and NB4.306 (2.6 fold decrease after 96hrs, p= 0.032). MA-21 was able to induce these effects by inducing significant G2/M cell cycle arrest and not by increased apoptosis or cellular differentiation. Cell cycle was assessed by Flow Cytometry after 96hrs of incubation and showed a significant increase in G2/M percentage in the ATRA sensitive and resistant cell lines compared to DMSO (NB4 cell line- 1.35 fold increase, p= 0.035; NB4.007/6- 1.35 fold increase, p= 0.015 and NB4.306- 2 fold increase, p= 0.023). Thus, we demonstrate novel synthetic methodology to synthesize boron containing novel retinoids and demonstrate the potential of these compounds as therapeutic agents in resistant leukemias.Figure1:Structures of ATRA and MA-21Figure1:. Structures of ATRA and MA-21 Disclosures: No relevant conflicts of interest to declare.

The Effect of Casein Kinase 2 Inhibition on three Leukemic Cell Lines

2020 ◽  
Vol 15 (3) ◽  
pp. 209-215
Author(s):  
Luka Horvat ◽  
Mariastefania Antica ◽  
Maja Matulić
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphocytic Leukaemia ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Notch Signalling ◽  
Leukaemia Cell ◽  
Leukaemia Cell Line

Background:: Casein Kinase 2 (CK2) is a Ser/Thr protein kinase that coregulates a great number of signalling pathways in the cell. It is involved in cell cycle regulation and cell proliferation, apoptosis, DNA damage response and gene transcription. Its substrates are numerous kinases and transcription factors. It was found to be upregulated in different tumours, and certain types of leukaemia are very sensitive to its inhibition. Objective:: We analysed the effects of casein kinase 2 inhibition on three leukaemia cell lines of B and T cell origin: Jurkat, a T cell line, CLL, a chronic B lymphocytic leukaemia cell line and 697, a pre-B acute lymphocytic leukaemia cell line. Besides cell proliferation and cytotoxicity analysis, the aim was to investigate the influence of CK2 inhibition on elements of the Notch signalling pathway. Notch signalling has an important role in blood cell differentiation, and CK2 regulates Ikaros, a tumour suppressor interfering with Notch signalling Methods:: and T leukaemia cells were treated with different concentrations of the CK2 inhibitor, CX-4945, for 6 days, and cell viability and proliferation were determined by Trypan Blue Exclusion Method. Analysis of gene expression was performed by RT-qPCR. Results:: All three cell lines were sensitive to CK2 inhibition and among them, 697 cells had two times lower IC50. In Jurkat and CLL cells changes in c-Myc and Notch pathway gene expression were found. Conclusion:: As CK2 is involved in numerous signalling circuits, we concluded that each cell type could have a cell-specific response in gene expression.

Identification and Expression Analysis of CD73 Inhibitors in Cervical Cancer

2020 ◽  
Vol 16 ◽  
Author(s):  
Jamshed Iqbal ◽  
Ayesha Basharat ◽  
Sehrish Bano ◽  
Syed Mobasher Ali Abid ◽  
Julie Pelletier ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Western Blot ◽  
Hela Cell ◽  
Cell Line ◽  
Cell Apoptosis ◽  
Immunofluorescence Staining ◽  
Cell Cycle Analysis ◽  
Hela Cell Line ◽  
Cell Line Hela

Aims: The present study was conducted to examine the inhibitory effects of synthesized sulfonylhydrazones on the expression of CD73 (ecto-5′-NT). Background: CD73 (ecto-5′-NT) represents the most significant class of ecto-nucleotidases which are mainly responsible for dephosphorylation of adenosine monophosphate to adenosine. Inhibition of CD73 played an important role in the treatment of cancer, autoimmune disorders, precancerous syndromes, and some other diseases associated with CD73 activity. Objective: Keeping in view the significance of CD73 inhibitor in the treatment of cervical cancer, a series of sulfonylhydrazones (3a-3i) derivatives synthesized from 3-formylchromones were evaluated. Methods: All sulfonylhydrazones (3a-3i) were evaluated for their inhibitory activity towards CD73 (ecto-5′-NT) by the malachite green assay and their cytotoxic effect was investigated on HeLa cell line using MTT assay. Secondly, most potent compound was selected for cell apoptosis, immunofluorescence staining and cell cycle analysis. After that, CD73 mRNA and protein expression were analyzed by real-time PCR and Western blot. Results: Among all compounds, 3h, 3e, 3b, and 3c were found the most active against rat-ecto-5′-NT (CD73) enzyme with IC50 (µM) values of 0.70 ± 0.06 µM, 0.87 ± 0.05 µM, 0.39 ± 0.02 µM and 0.33 ± 0.03 µM, respectively. These derivatives were further evaluated for their cytotoxic potential against cancer cell line (HeLa). Compound 3h and 3c showed the cytotoxicity at IC50 value of 30.20 ± 3.11 µM and 86.02 ± 7.11 µM, respectively. Furthermore, compound 3h was selected for cell apoptosis, immunofluorescence staining and cell cycle analysis which showed promising apoptotic effect in HeLa cells. Additionally, compound 3h was further investigated for its effect on expression of CD73 using qRT-PCR and western blot. Conclusion: Among all synthesized compounds (3a-3i), Compound 3h (E)-N'-((6-ethyl-4-oxo-4H-chromen-3-yl) methylene)-4-methylbenzenesulfonohydrazide was identified as most potent compound. Additional expression studies conducted on HeLa cell line proved that this compound successfully decreased the expression level of CD73 and thus inhibiting the growth and proliferation of cancer cells.

scholarly journals A New CDK2 Inhibitor with 3-Hydrazonoindolin-2-One Scaffold Endowed with Anti-Breast Cancer Activity: Design, Synthesis, Biological Evaluation, and In Silico Insights

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 412
Author(s):  
Mohammad M. Al-Sanea ◽  
Ahmad J. Obaidullah ◽  
Mohamed E. Shaker ◽  
Garri Chilingaryan ◽  
Mohammed M. Alanazi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cell Line ◽  
Inhibitory Activity ◽  
Flow Cytometric Analysis ◽  
Docking Studies ◽  
Biological Evaluation ◽  
Intrinsic Activity ◽  
Binding Interaction ◽  
Mcf 7

Background: Cyclin-dependent kinases (CDKs) regulate mammalian cell cycle progression and RNA transcription. Based on the structural analysis of previously reported CDK2 inhibitors, a new compound with 3-hydrazonoindolin-2-one scaffold (HI 5) was well designed, synthesized, and biologically evaluated as a promising anti-breast cancer hit compound. Methods: The potential anti-cancerous effect of HI 5 was evaluated using cytotoxicity assay, flow cytometric analysis of apoptosis and cell cycle distribution, ELISA immunoassay, in vitro CDK2/cyclin A2 activity, and molecular operating environment (MOE) virtual docking studies. Results: The results revealed that HI 5 exhibits pronounced CDK2 inhibitory activity and cytotoxicity in human breast cancer MCF-7 cell line. The cytotoxicity of HI 5 was found to be intrinsically mediated apoptosis, which in turn, is associated with low Bcl-2 expression and high activation of caspase 3 and p53. Besides, HI 5 blocked the proliferation of the MCF-7 cell line and arrested the cell cycle at the G2/M phase. The docking studies did not confirm which one of geometric isomers (syn and anti) is responsible for binding affinity and intrinsic activity of HI 5. However, the molecular dynamic studies have confirmed that the syn-isomer has more favorable binding interaction and thus is responsible for CDK2 inhibitory activity. Discussion: These findings displayed a substantial basis of synthesizing further derivatives based on the 3-hydrazonoindolin-2-one scaffold for favorable targeting of breast cancer.

scholarly journals Effect of Aspirin on Cell Growth of Human MG-63 Osteosarcoma Line

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
E. De Luna-Bertos ◽  
J. Ramos-Torrecillas ◽  
O. García-Martínez ◽  
L. Díaz-Rodríguez ◽  
C. Ruiz
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Line ◽  
Bone Tissue ◽  
Tissue Repair ◽  
Mg63 Cell ◽  
Pharmacological Action ◽  
Inhibitory Effect ◽  
Cell Cycle Analysis ◽  
Inflammatory Drugs

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in bone tissue repair treatment for their pharmacological action. The objective of this study was to determine the effect of aspirin, on osteoblast growth, using MG63 cell line as osteoblast model. MTT spectrophotometry results showed that 20, 100, and 1000 μM aspirin doses have an inhibitory effect on growth. Cell cycle analysis revealed that aspirin doses of 100 and 1000 μM arrest the cell cycle in phase GO/G1. Parallel apoptosis/necrosis studies showed no changes in comparison to control cells after treatment with 1 or 10 μM aspirin but a significantly increased percentage of cells in apoptosis at doses of 20, 100, and 1000 μM. We highlight that treatment of osteoblast-like cells with 1000 μM aspirin increased not only the percentage of cells in apoptosis but also the percentage of necrotic cells, which was not observed in aspirin treatments at lower doses.

Cell cycle analysis during retinoic acid induced differentiation of a human embryonal carcinoma-derived cell line

1987 ◽  
Vol 20 (2-3) ◽  
pp. 153-160 ◽  
Author(s):  
Christine L. Mummery ◽  
Marga A. van Rooijen ◽  
Stieneke E. van den Brink ◽  
Siegfried W. de Laat
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Cell Line ◽  
Embryonal Carcinoma ◽  
Cell Cycle Analysis ◽  
Induced Differentiation

scholarly journals Preclinical Activity of the MPS1 Inhibitor S81694 in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2631-2631
Author(s):  
Anna Kaci ◽  
Emilie Adiceam ◽  
Melanie Dupont ◽  
Marine Garrido ◽  
Jeannig Berrou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Solid Tumors ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Jurkat Cells ◽  
Cell Cycle Distribution ◽  
Monopolar Spindle

Introduction: The dual-specificity protein kinase, monopolar spindle 1 (Mps1) is one the main kinases of the spindle assembly checkpoint (SAC) critical for accurate segregation of sister chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. Mps1 is known to be overexpressed in several solid tumors including triple negative breast cancer. Thus, Mps1 seems to be a promising target and small molecules targeting Mps1 entered clinical trials in solid tumors. ALL originates from malignant transformation of B-and T-lineage lymphoid precursors with a variety of genetic aberrations including chromosome translocations, mutations, and aneuploidies in genes responsible for cell cycle regulation and lymphoid cell development. While outcome is excellent for pediatric patients and younger adults, relapsed and refractory disease still remain a clinical challenge for elder patients. Here, we demonstrate for the first time preclinical efficacy of the small molecule Mps1 inhibitor (Mps1i) S81694 in T- and B- ALL cells including BCR-ABL1+-driven B-ALL. Materials and Methods: Expression of Mps1 was determined by RT-qPCR and WB in JURKAT, RS4-11 and BCR-ABL1+ cells (BV-173 and TOM-1). A small molecule Mps1i (S81694) was tested alone (0 to 1000nM) or in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1+ ALL cell lines. Cell viability and IC50 was assessed by MTS assays after exposure to Mps1i for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Bliss model. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of Mps1i. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation at 48h. Phosphorylation of Mps1 was detected in synchronized (by nocodazole and MG-132) cells by immunofluorescence using an anti phospho-Mps1 antibody detecting Thr33/Ser37 residues. Time-lapse microscopy was used in cell lines in presence or absence of S81694 to determine mitosis duration. Bone marrow (BM) nucleated patient cells were obtained after informed consent and incubated in methylcellulose with cytokines with or without Mps1i for 2 weeks to determine colony growth. Results: Expression of Mps1 could be detected by RT-qPCR and at the protein level by WB in all cell lines (Figure 1A and B ). IC50 after Mps1i exposure alone was 126nM in JURKAT cells, 51nM in RS4-11 cells, 75nM in BV-173 cells and 83nM in TOM-1. Significant apoptosis as detected by phosphatidylserine exposure and PI incorporation in all cell lines with BCR-ABL1+ cell lines BV-173 and TOM-1 cells being the most sensitive (80% and 60% apoptotic cells respectively)(Figure 1C). Upon Mps1i exposure we observed targeted inhibition of Mps1 phosphorylation at Thr33/Ser37 residues indicating the specific on target effect of S81694 by inhibiting Mps1 autophosphorylation (Figure 1D and E). Cell cycle profile was generally lost after treatment with S81694 in all cell lines indicating aberrant 2n/4n distribution due to SAC abrogation (Figure 1F). Furthermore, we demonstrated that S81694 exposure accelerated significantly mitosis in BV-173 cell line from 36 minutes to 19 minutes indicating effective inhibition of SAC function (Figure 1G). Interestingly, S81694 induced significant apoptosis (70%) in the imatinib resistant BV173 cell line bearing the E255K-BCR-ABL1-mutation. Combination of S81694 with TKI imatinib, dasatinib and nilotinib (but not ponatinib) was strongly synergistic in BCR-ABL1+ cells (Figure 1H). Finally, we observed inhibition of colony formation in a patient with BCR-ABL1+ B-ALL after exposure to 100nM and 250nM S81694 (reduction of 85% and 100% respectively)(Figure 1I). Conclusion: Mps1i S81694 yields significant preclinical activity in T-and B-cell ALL including BCR-ABL1+ models. Interestingly S81694 was efficacious in a TKI resistant cell line. Disclosures Kaci: Institut de Recherches Internationales Servier (IRIS): Employment. Garrido:Institut de Recherches Internationales Servier (IRIS): Employment. Burbridge:Institut de Recherches Internationales Servier (IRIS): Employment. Dombret:AGIOS: Honoraria; CELGENE: Consultancy, Honoraria; Institut de Recherches Internationales Servier (IRIS): Research Funding. Braun:Institut de Recherches Internationales Servier (IRIS): Research Funding.

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.

scholarly journals A Novel Analysis Method for Evaluating the Interplay of Oxygen and Ionizing Radiation at the Gene Level

2021 ◽  
Vol 12 ◽  
Author(s):  
Jeannette Jansen ◽  
Patricia Vieten ◽  
Francesca Pagliari ◽  
Rachel Hanley ◽  
Maria Grazia Marafioti ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Radiation Treatment ◽  
Hierarchical Cluster ◽  
Specific Gene ◽  
Analysis Method ◽  
Whole Genome Analysis ◽  
Gene Level ◽  
The Impact

Whilst the impact of hypoxia and ionizing radiations on gene expression is well-understood, the interplay of these two effects is not. To better investigate this aspect at the gene level human bladder, brain, lung and prostate cancer cell lines were irradiated with photons (6 Gy, 6 MV LINAC) in hypoxic and normoxic conditions and prepared for the whole genome analysis at 72 h post-irradiation. The analysis was performed on the obtained 20,000 genes per cell line using PCA and hierarchical cluster algorithms to extract the most dominant genes altered by radiation and hypoxia. With the help of the introduced novel radiation-in-hypoxia and oxygen-impact profiles, it was possible to overcome cell line specific gene regulation patterns. Based on that, 37 genes were found to be consistently regulated over all studied cell lines. All DNA-repair related genes were down-regulated after irradiation, independently of the oxygen state. Cell cycle-dependent genes showed up-regulation consistent with an observed change in cell population in the S and G2/M phases of the cell cycle after irradiation. Genes behaving oppositely in their regulation behavior when changing the oxygen concentration and being irradiated, were immunoresponse and inflammation related genes. The novel analysis method, and by consequence, the results presented here have shown how it is important to consider the two effects together (oxygen and radiation) when analyzing gene response upon cancer radiation treatment. This approach might help to unrevel new gene patterns responsible for cancer radioresistance in patients.

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