RSK Inhibition Suppresses AML Proliferation through Activation of DNA Damage Pathways and S Phase Arrest

Abstract The 90 kDa Ribosomal S6 Kinase (RSK), downstream of the ERK signaling pathway, has recently been implicated in a wide variety of cancers, ranging from lung cancer to medulloblastoma, as a driver of cancer cell proliferation and survival. However, its role in Acute Myeloid Leukemia (AML) remains unknown. Thus, the goal of this study was to characterize RSK-dependent signaling pathways in AML, with the overall hypothesis that disruption of this pathway represents a potential strategy for the treatment of AML. The RSK family consists of four gene isoforms, RSK1-4 (RPS6KA1 (RSK1), RPS6KA2 (RSK3), RPS6KA3 (RSK2), RPS6KA4 (RSK4). Knockdown (KD) of RSK1 by shRNA in HL-60 and KG-1 cell lines resulted in reduced AML cell growth in vitro. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were injected with 2x106 HL-60 or KG-1 RSK1KD cells and vector control transduced cells in order to investigate the effects of RSK1 KD on AML cell growth and survival in vivo. Mice injected with RSK1 KD cells exhibited prolonged survival by 17 and 21 days respectively for HL-60 and KG-1 cell induced disease (p=0.0023 and 0.0018 respectively). These data indicate that RSK1 knockdown inhibits leukemia progression, and RSK1 is required for maximal proliferation of AML cells in vivo. Pharmacological inhibition of total RSK (RSK1-4) by the small molecule inhibitor BI-D1870 reduced AML cell growth and induced cell death in both AML cell lines and patient samples after treatment for 48 hours. The IC50 for growth inhibition was 1.8 uM for MOLM-13, 1.6 uM for MV-4-11, and 1.9 uM for HL-60 cells. In methylcellulose colony assays, normal hematopoietic stem and progenitor cell proliferation was not affected by RSK inhibition up to a concentration of 15 uM, establishing an approximately 10-fold therapeutic index. To elucidate the mechanism by which RSK inhibition suppresses AML proliferation, we performed cell cycle analysis with HL-60 cells. RSK inhibition by BI-D1870 resulted in delayed S-phase progression and accumulation of cells in late S-phase with increased pH2AX, cPARP, and CDK2/Cyclin A expression, as measured by flow cytometry. These data indicate that inhibition of RSK leads to activation of DNA damage pathways and arrest in S-phase, resulting in apoptosis. Inhibition of CDK activity rescued S-phase arrest, demonstrating that activation and dysregulation of CDK are crucial mediators of RSK inhibitor-induced S-phase arrest. In summary, this is the first study to demonstrate that RSK plays an important role in maintaining AML cell survival and proliferation and to position RSK as a promising target for treatment of AML. Disclosures No relevant conflicts of interest to declare.

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Combined Inhibition of Wee1 and Poly(ADP Ribose) Polymerase1/2 Synergistically Inhibits Acute Myeloid Leukemia Cells By Enhancing DNA Damage and the Induction of Apoptosis

Blood ◽

10.1182/blood.v124.21.3621.3621 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3621-3621 ◽

Cited By ~ 1

Author(s):

Jonathan C Snedeker ◽

Tamara M Burleson ◽

Raoul Tibes ◽

Christopher C. Porter

Keyword(s):

Flow Cytometry ◽

Cell Proliferation ◽

Dna Damage ◽

Cell Lines ◽

Conflicts Of Interest ◽

Combination Index ◽

Annexin V ◽

Concomitant Treatment ◽

Western Blots

Abstract Introduction: Successful treatment of AML remains dependent upon cytotoxic chemotherapy. However, traditional regimens are not well tolerated by older patients who are at highest risk of disease, and salvage rates after relapse are low, necessitating novel therapeutic strategies. Our groups identified Wee1 as a potential therapeutic target in AML, particularly in the context of concomitant treatment with cytarabine (Tibes et al, Blood, 2012; Porter et al, Leukemia, 2012). Wee1 inhibits CDK1&2 via phosphorylation thereby stalling cell cycle progression. One consequence of Wee1 inhibition/CDK1 activation is impairment of DNA repair via homologous recombination (Krajewska et al, Oncogene, 2013). Cells in which HR is impaired are dependent upon Parp1/2 function, and HR deficient cells are particularly sensitive to Parp1/2 inhibition. Therefore, we hypothesized that combined Wee1 and Parp1/2 inhibition may result in greater inhibition of AML cell proliferation and survival than either alone. Methods: Human AML cell lines, MV4-11 and Molm-13, and a mouse AML that expresses MLL-ENL/FLT3-ITD were cultured with various concentrations of a Wee1 inhibitor (AZ1775) and a Parp1/2 inhibitor (olaparib) and counted 72 hours later by propidium iodide exclusion and flow cytometry. In some experiments, cells were split into fresh media to recover for 72 more hours. Combination Index (CI) values were calculated by the method of Chou and Talalay. Apoptosis was measured using Annexin V/7AAD and flow cytometry. Western blots were used to confirm inhibition of CDK1/2 phosphorylation and to measure DNA damage induction (gamma-H2AX). Results: Combined inhibition of Wee1 and Parp1/2 was synergistic, as measured by cell numbers at 72 hours, in all 3 cell lines tested, with combination index values ranging from 0.3 to 0.9. When cells were allowed to recover after treatment, those treated by single agents were able to continue proliferating. However, those treated with the combination did not recover as well or at all, indicating greatly impaired proliferative capacity. Combined inhibition of Wee1 and Parp1/2 also resulted in a significant increase in apoptosis greater than either drug alone. Western blots for gamma-H2AX confirmed that the combination of Wee1 and Parp1/2 resulted in more DNA damage than either drug alone. Discussion: Combined inhibition of Wee1 and Parp1/2 results in greater inhibition of AML cell proliferation, DNA damage and apoptosis than either drug alone. Future studies will include experiments with primary patient samples, as well as in vivo trials combining Wee1 inhibition with Parp1/2 inhibition. These preliminary studies raise the possibility of rational combinations of targeted agents for leukemia in those for whom conventional chemotherapeutics may not be well tolerated. Disclosures No relevant conflicts of interest to declare.

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Selenocystine inhibits JEG-3 cell growth in vitro and in vivo by triggering oxidative damage-mediated S-phase arrest and apoptosis

Journal of Cancer Research and Therapeutics ◽

10.4103/jcrt.jcrt_864_17 ◽

2018 ◽

Vol 14 (7) ◽

pp. 1540 ◽

Cited By ~ 3

Author(s):

Zhigang Wei ◽

Ming Zhao ◽

Yajun Hou ◽

Xiaoting Fu ◽

Dawei Li ◽

...

Keyword(s):

Cell Growth ◽

Oxidative Damage ◽

S Phase ◽

Phase Arrest ◽

S Phase Arrest

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Variations in Mre11/Rad50/Nbs1 status and DNA damage-induced S-phase arrest in the cell lines of the NCI60 panel

BMC Cancer ◽

10.1186/1471-2407-11-206 ◽

2011 ◽

Vol 11 (1) ◽

Cited By ~ 7

Author(s):

Kristen M Garner ◽

Alan Eastman

Keyword(s):

Dna Damage ◽

Cell Lines ◽

S Phase ◽

Phase Arrest ◽

S Phase Arrest ◽

Nci60 Panel

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Water-soluble oxoglaucine-Y(iii), Dy(iii) complexes: in vitro and in vivo anticancer activities by triggering DNA damage, leading to S phase arrest and apoptosis

Dalton Transactions ◽

10.1039/c5dt00926j ◽

2015 ◽

Vol 44 (25) ◽

pp. 11408-11419 ◽

Cited By ~ 15

Author(s):

Jian-Hua Wei ◽

Zhen-Feng Chen ◽

Jiao-Lan Qin ◽

Yan-Cheng Liu ◽

Zhu-Quan Li ◽

...

Keyword(s):

Dna Damage ◽

Anticancer Activity ◽

S Phase ◽

Water Soluble ◽

Anticancer Activities ◽

Phase Arrest ◽

S Phase Arrest

The complexes exhibited considerable in vitro and in vivo anticancer activity, and higher safety than ciplatin.

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Telmisartan Inhibits Cell Proliferation and Tumor Growth of Esophageal Squamous Cell Carcinoma by Inducing S-Phase Arrest In Vitro and In Vivo

International Journal of Molecular Sciences ◽

10.3390/ijms20133197 ◽

2019 ◽

Vol 20 (13) ◽

pp. 3197 ◽

Cited By ~ 5

Author(s):

Takanori Matsui ◽

Taiga Chiyo ◽

Hideki Kobara ◽

Shintaro Fujihara ◽

Koji Fujita ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Squamous Cell Carcinoma ◽

Esophageal Squamous Cell Carcinoma ◽

Tumor Growth ◽

S Phase ◽

Phase Arrest ◽

S Phase Arrest

Esophageal squamous cell carcinoma (ESCC) is the most common primary esophageal malignancy. Telmisartan, an angiotensin II type 1 (AT1) receptor blocker (ARB) and a widely used antihypertensive, has been shown to inhibit proliferation of various cancer types. This study evaluated the effects of telmisartan on human ESCC cell proliferation in vitro and in vivo and sought to identify the microRNAs (miRNAs) involved in these antitumor effects. We examined the effects of telmisartan on three human ESCC cell lines (KYSE150, KYSE180, and KYSE850). Telmisartan inhibited proliferation of these three cell lines by inducing S-phase arrest, which was accompanied by decreased expression of cyclin A2, cyclin-dependent kinase 2, and other cell cycle-related proteins. Additionally, telmisartan reduced levels of phosphorylated ErbB3 and thrombospondin-1 in KYSE180 cells. Furthermore, expression of miRNAs was remarkably altered by telmisartan in vitro. Telmisartan also inhibited tumor growth in vivo in a xenograft mouse model. In conclusion, telmisartan inhibited cell proliferation and tumor growth in ESCC cells by inducing cell-cycle arrest.

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Copper complexes as prospective anticancer agents: in vitro and in vivo evaluation, selective targeting of cancer cells by DNA damage and S phase arrest

RSC Advances ◽

10.1039/c8ra00954f ◽

2018 ◽

Vol 8 (30) ◽

pp. 16973-16990 ◽

Cited By ~ 21

Author(s):

Dharmasivam Mahendiran ◽

Sethu Amuthakala ◽

Nattamai S. P. Bhuvanesh ◽

Raju Senthil Kumar ◽

Aziz Kalilur Rahiman

Keyword(s):

Dna Damage ◽

Anticancer Agents ◽

Copper Complexes ◽

S Phase ◽

In Vivo Evaluation ◽

Phase Arrest ◽

Selective Targeting ◽

S Phase Arrest

The thiosemicarbazone-based copper(i) complexes causing S phase arrest and apoptosis involving the mitochondrial controlled pathway has been investigated.

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Expression of 1N3R-Tau Isoform Inhibits Cell Proliferation by Inducing S Phase Arrest in N2a Cells

PLoS ONE ◽

10.1371/journal.pone.0119865 ◽

2015 ◽

Vol 10 (3) ◽

pp. e0119865 ◽

Cited By ~ 4

Author(s):

Li Li ◽

Zhi-Peng Xu ◽

Gong-Ping Liu ◽

Cheng Xu ◽

Zhi-Hao Wang ◽

...

Keyword(s):

Cell Proliferation ◽

S Phase ◽

Phase Arrest ◽

N2a Cells ◽

S Phase Arrest ◽

Tau Isoform

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Dysregulation of hsa-miR-34a and hsa-miR-449a leads to overexpression of PACS-1 and loss of DNA damage response (DDR) in cervical cancer

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014048 ◽

2020 ◽

Vol 295 (50) ◽

pp. 17169-17186

Author(s):

Mysore S. Veena ◽

Santanu Raychaudhuri ◽

Saroj K. Basak ◽

Natarajan Venkatesan ◽

Parameet Kumar ◽

...

Keyword(s):

Cell Cycle ◽

Cervical Cancer ◽

Dna Damage ◽

Cell Growth ◽

Cell Lines ◽

Cancer Cell ◽

Dna Damage Response ◽

S Phase ◽

Cervical Cancer Cell ◽

Damage Response

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3′ terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys382-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.

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