scholarly journals Inhibition of ATR Overcomes Chemotherapy Resistance in p53 Deficient Myeloma Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3109-3109
Author(s):  
Louise Bouard ◽  
Benoit Tessoulin ◽  
Géraldine Descamps ◽  
Cyrille Touzeau ◽  
Philippe Moreau ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Dna Repair ◽  
Chemotherapy Resistance ◽  
S Phase ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Myeloma Cells ◽  
Strand Breaks ◽  
Cycle Arrest

In MM, as well as in most hematological malignancies, deficiency in p53 pathway (mainly because of TP53 deletion and/or mutation) is associated with resistance to treatments (Tessoulin Blood Reviews 2017; 31:251). Recent clinical studies have shown that deletion or mutation of TP53 are the most adverse prognostic values for patients (Thakurta Blood 2019;133:1217). Although these patients are easily identified, there is no dedicated therapies for them. p53 pathway is central for homeostasis and cell adaptation/response to many stresses, including DNA repair orchestration and survival regulation. In p53 deficient cells, DNA damaging drugs don't induce massive apoptosis and cells escape to death. In normal cells, DNA damaging drugs induce cell cycle arrest and DNA repair, mainly orchestrated by p53 target genes. Cell cycle arrest in S phase, which is critical for allowing homologous DNA repair, is activated by cell cycle check-point inhibitor such as Chk1, an ATR target. In p53 deficient cells, inhibiting check point inhibitor using ATR inhibitor should allow DNA damaged cells to progress into cell cycle despite the lack of repair and in fine induce replicative/mitotic catastrophe. The aim of this study was to assess whether inhibiting ATR in p53 deficient myeloma cells could overcome chemotherapy resistance. ATR inhibitor, VE-821, was assessed in 13 human myeloma cell lines (HMCLs) alone and in combination with DNA damaging agents, CX5461, a G quadruplex inhibitor, or melphalan, the « myeloma » alkylating drug. The HMCL cohort included 8 HMCLs, 5 TP53Abn and 5 TP53wt. Cell viability was assessed using Cell Titer Glo assay or using flow cytometry (loss of AnnexinV or CD138 staining in HMCLs or primary myeloma cells, respectively). In our cohort of 13 HMCLs and by contrast to previous results, CX5461 was more efficient in TP53wt than in TP53abn HMCLs (mean of death at 0.5mM was 43% versus 24%, p=0.04). Melphalan was also more potent in TP53wt than in TP53abn HMCLs (LD50 values were 26 mM versus 10 mM, p=0.008). By contrast, ATR inhibitor VE-821 (2.5mM) was efficient in both types of HMCLs (mean of death in TP53wt was 45% and 28% in TP53abn HMCLs, p=0.6). Combination of CX5641 (0.5mM) with VE-821 (2.5mM) was more efficient than each drug alone and efficacy was not dependent on TP53 status (mean of death in TP53wt was 69% versus 56% in TP53abn HMCLs, p=0.6): interestingly, combo was efficient in all TP53abn HMCLs, being either additive (n=5) or even synergistic (n=3). By contrast, combo was not efficient in all TP53wt HMCLs (either additive or antagonist). Combination of melphalan (10 mM) with VE-821 (2.5mM) was also synergistic in TP53abn HMCLs (mean of cell death was 9% with melphalan and 73% for combo, p<0.05). Preliminary results of combos in 6 consecutive primary samples with MM or plasma cell leukemia (3 TP53wt and 3 TP53abn) demonstrated efficacy. Indeed, in the 3 TP53abn samples, both CX5641/VE-821 and melphalan/VE-821 combos displayed synergism or additivity: median of expected values versus observed values was 61% versus 74% for CX5641/VE-821, and 98% versus 89% for melphalan/VE-821, respectively. In the 3 TP53wt samples, combos displayed additivity or antagonism: median of expected versus observed values was 15% versus 15% for CX5641/VE-821, and 100% versus 62% for melphalan/VE-821, respectively. In normal peripheral blood cells (n=2), both combos were not cytotoxic (mean values of cell death were 0% with CX5641/VE-821 and 3% with melphalan/VE-821). To decipher the molecular pathway involved in cell response, we monitored cell cycle using BrdU/IP assay, replicative stress response using Chk1 phosphorylation and DNA double strand breaks using Comets assays in 3 TP53abn HMCLs. At 24h, CX5641 induced an increase of cells in S (mean of increase 12%) and G2M phases (11%), while VE-821 didn't modify cell cycle. Combination of CX5641 with VE-821 induced a dramatic increase of cells in G2M (20%) (and in subG2 phase), and a decrease of cells in S phase (10%), indicating that cells blocked in S phase by CX5641 were released by VE-821.CX5641 induced Chk1 phosphorylation, which was inhibited by addition of VE-821, confirming the CX5641/ATR/Chk1 signaling. Finally, CX5641 and VE-821 induced comets, confirming irreversible DNA double strand breaks. All these results show that inhibition of ATR after inducing DNA damage in TP53abn myeloma cells efficiently induces cell death, while preserving normal cells. Disclosures Moreau: Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

ANKHD1 Silencing Delays S Phase Progression in Multiple Myeloma Cells Via Activation of ATM/ATR -CDC25a Pathway

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5624-5624
Author(s):  
Dhyani Anamika ◽  
Patricia Favaro ◽  
Sara Teresinha Olalla Saad
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Dna Damage ◽  
S Phase ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Myeloma Cells ◽  
Checkpoint Kinase ◽  
Strand Breaks ◽  
Phase Progression

Abstract Ankyrin repeat and KH domain-containing protein 1, ANKHD1, is highly expressed in myeloma cells and plays an important role in multiple myeloma (MM) progression and growth. ANKHD1 is found to be overexpressed in S phase of cell cycle in MM cells and silencing of ANKHD1 expression leads to accumulation of cells in S phase, suggesting a role in S phase progression (1). Earlier studies by our group reported that ANKHD1 silencing downregulates all replication dependent histones and that this downregulation may be associated with replication stress and DNA damage (2). We observed increased expression of γH2AX protein (phosphorylated histone H2A variant, H2AX, at Serine 139), a marker for DNA double strand breaks (DSBs) and an early sign of DNA damage induced by replication stress, in ANKHD1 silenced MM cells. In the present study we further sought to investigate the mechanisms underlying the induction of DNA damage on ANKHD1 silencing. We first confirmed the increased expression of γH2AX by flow cytometry analysis and observed that both the mean fluorescence intensity as well as percentage of γH2AX positive cells were higher in ANKHD1 silenced MM cells as compared to control cells. Phosphorylation of histone 2AX requires activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated)andATR (ATM-Rad3-related) that serves as central components of the signaling cascade initiated by DSBs. Hence, we checked for the expression of these kinases and observed increased phosphorylation of both ATM and ATR kinases in ANKHD1 silenced MM cells. There was no difference in the expressions of DNA-PKcs in control and ANKHD1 silenced cells by western blot. We next checked for the expression of CHK1 (checkpoint kinase 1) and CHK2 (checkpoint kinase 2), essential serine threonine kinases downstream of ATM and ATR. We observed a decrease in pCHK2 (phosphorylated CHK2 at Thr 68), with no change in expression of pCHK1 (phosphorylated CHK1 at Ser 345) total CHK1 or total CHK2. We also checked for expression of CDC25a (a member of the CDC25 family of dual-specificity phosphatases), that is specifically degraded in response to DNA damage (DSBs) and delays S phase progression via activation of ATM /ATR-CHK2 signaling pathway. Expression of CDC25a was significantly decreased in ANKHD1 silencing cells, confirming the induction of DSBs, and probably accounting for S phase delay on ANKHD1 silencing. Since there was decrease in active CHK2 (pCHK2) and no change in CHK1 required for degradation of CDC25a, we assume that decrease in CDC25a in ANKHD1 silenced MM cells may be via activation of ATM/ ATR pathway independent of CHK2/CHK1. Expression of several other downstream factors of DSBs induced DNA damage response and repair such as BRCA1, PTEN, DNMT1, SP1, HDAC2 were also found to be modulated in ANKHD1 silenced MM cells. In conclusion, ANKHD1 silencing in MM cells leads to DNA damage and modulates expression of several genes implicated in DNA damage and repair. DNA damage induced after ANKHD1 silencing in MM cells activates ATM/ ATR-CDC25a pathway which may lead to the activation of S phase checkpoint in MM cells. Results however are preliminary and further studies are required to understand the role of ANKHD1 in intra S phase check point. References: 1) ANKHD1 regulates cell cycle progression and proliferation in multiple myeloma cells. Dhyani et al. FEBS letters 2012; 586: 4311-18. 2) ANKHD1 is essential for repair of DNA double strand breaks in multiple myeloma. Dhyani et al. ASH Abstract, Blood 2015; 126:1762. Disclosures No relevant conflicts of interest to declare.

High NaCl causes Mre11 to leave the nucleus, disrupting DNA damage signaling and repair

2003 ◽  
Vol 285 (2) ◽  
pp. F266-F274 ◽  
Author(s):  
Natalia I. Dmitrieva ◽  
Dmitry V. Bulavin ◽  
Maurice B. Burg
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
Damage Response

High NaCl causes DNA double-strand breaks and cell cycle arrest, but the mechanism of its genotoxicity has been unclear. In this study, we describe a novel mechanism that contributes to this genotoxicity. The Mre11 exonuclease complex is a central component of DNA damage response. This complex assembles at sites of DNA damage, where it processes DNA ends for subsequent activation of repair and initiates cell cycle checkpoints. However, this does not occur with DNA damage caused by high NaCl. Rather, following high NaCl, Mre11 exits from the nucleus, DNA double-strand breaks accumulate in the S and G2 phases of the cell cycle, and DNA repair is inhibited. Furthermore, the exclusion of Mre11 from the nucleus by high NaCl persists following UV or ionizing radiation, also preventing DNA repair in response to those stresses, as evidenced by absence of H2AX phosphorylation at places of DNA damage and by impaired repair of damaged reporter plasmids. Activation of chk1 by phosphorylation on Ser345 generally is required for DNA damage-induced cell cycle arrest. However, chk1 does not become phosphorylated during high NaCl-induced cell cycle arrest. Also, high NaCl prevents ionizing and UV radiation-induced phosphorylation of chk1, but cell cycle arrest still occurs, indicating the existence of alternative mechanisms for the S and G2/M delays. DNA breaks that occur normally during processes such as DNA replication and transcription, as well as damages to DNA induced by genotoxic stresses, ordinarily are rapidly repaired. We propose that inhibition of this repair by high NaCl results in accumulation of DNA damage, accounting for the genotoxicity of high NaCl, and that cell cycle delay induced by high NaCl slows accumulation of DNA damage until the DNA damage-response network can be reactivated.

scholarly journals PS2 - 153 Dianhydrogalactitol (VAL-083) Treatment Causes Irreparable DNA Double-Strand Breaks, S/G2 Phase Cell-Cycle Arrest and Cell Death in Cancer Cells

2016 ◽  
Vol 43 (S4) ◽  
pp. S12-S13
Author(s):  
B. Zhai ◽  
A. Steino ◽  
J. Bacha ◽  
D. Brown ◽  
M. Daugaard
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Death ◽  
Cell Cycle Arrest ◽  
Cancer Cells ◽  
Double Strand Breaks ◽  
Phase Cell ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cycle Arrest

Dianhydrogalactitol (VAL-083) is a unique bi-functional alkylating agent causing N7-guanine-methylation and inter-strand DNA crosslinks. VAL-083 readily crosses the blood-brain barrier, accumulates in brain tumor tissue and has shown activity in prior NCI-sponsored clinical trials against various cancers, including glioblastoma (GBM) and medulloblastoma. VAL-083 is also active against GBM cancer stem cells and acts as a radiosensitizer independent of O6-methylguanine-DNA methyltransferase activity (in contrast to e.g. temozolomide and BCNU). Here we report new insights into VAL-083 mechanism of action by showing that VAL-083 induces irreversible cell-cycle arrest and cell death caused by replication-dependent DNA damage. In lung (H2122, H1792, H23, A549) and prostate (PC3, LNCaP) cancer cell lines VAL-083 treatment caused irreversible S/G2 cell-cycle arrest and cell death (IC50 range 3.06-25.7 µM). VAL-083 pulse-treatment led to persistent phosphorylation of DNA double-strand breaks (DSB) sensors ATM, single-strand DNA-binding Replication Protein A (RPA32), and histone variant H2A.X, suggesting persistent DNA lesions. After 10 months in culture with increasing VAL-083 concentrations, H1792 and LNCaP cells survive at concentrations up to 9.4 µM and 7.4 µM, respectively, suggesting that efficient resistance mechanisms are not easily acquired by the cancer cells. Taken together with previous results showing that VAL-083 circumvents cisplatin-resistance and is less dependent on p53 activity than cisplatin, these results suggest a molecular mechanism for VAL-083 that differs from both TMZ, BCNU and cisplatin. They further suggest that irreparable DNA damage induced by VAL-083 is impervious to common strategies employed by cancer cells to escape effects of alkylating drugs used in GBM treatment.

scholarly journals SET8 Is a Potential Therapeutic Target in MM

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4435-4435
Author(s):  
Herviou Laurie ◽  
Fanny Izard ◽  
Elke De Bruyne ◽  
Eva Desmedt ◽  
Anqi Ma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Myeloma Cells ◽  
Strand Breaks ◽  
Damage Response

Abstract Epigenetic regulation mechanisms - such as histone marks, DNA methylation and miRNA - are often misregulated in cancers and are associated with tumorigenesis and drug resistance. Multiple Myeloma (MM) is a malignant plasma cell disease that accumulates within the bone marrow. Epigenetic modifications in MM are associated not only with cancer development and progression, but also with resistance to chemotherapy. This epigenetic plasticity can be targeted with epidrugs, nowadays used in treatment of several cancers. We recently identified a significant overexpression of the lysine histone methyltransferase SETD8 in MM cells (HMCLs; N=40) compared with normal plasma cells (N=5) (P<0.001). SETD8 (also known as SET8, PR-Set7, KMT5A) is the sole enzyme responsible for the monomethylation of histone H4 at lysine 20 (H4K20me1) which has been linked to chromatin compaction and cell-cycle regulation. In addition, SETD8 induces the methylation of non-histone proteins, such as the replication factor PCNA, the tumor suppressor P53 and its stabilizing protein Numb. While SETD8-mediated methylation of P53 and Numb inhibits apoptosis, PCNA methylation upon SETD8 enhances the interaction with the Flap endonuclease FEN1 and promotes cancer cell proliferation. SETD8 is also implicated in DNA damage response, helping 53BP1 recruitment at DNA double-strand breaks. Consistent with this, overexpression of SETD8 is found in various types of cancer and has been directly implicated in breast cancer invasiveness and metastasis. A role of SETD8 in development of MM has however never been described. We found that high SETD8 expression is associated with a poor prognosis in 2 independent cohorts of newly diagnosed patients (UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N=158). Specific SETD8 inhibition with UNC-0379 inhibitor, causing its degradation and H4K20me1 depletion, leads to significant growth inhibition of HMCLs (N=10) and the murine cell lines 5T33MM and 5TGM1. MM cells treated with UNC-0379 presented a G0/G1 cell cycle arrest after 24h of treatment, followed by apoptosis 48h later. To confirm that SETD8 inhibition is as efficient on primary MM cells from patients, primary MM cells (N=8) were co-cultured with their bone marrow microenvironment and recombinant IL-6 and treated for 4 days with UNC-0379. Interestingly, treatment of MM patient samples with UNC-0379 reduces the percentage of myeloma cells (65%; P<0.005) without significantly affecting the non-myeloma cells, suggesting a specific addiction of primary myeloma cells to SETD8 activity. Melphalan is an alkylating agent commonly used in MM treatment. As SETD8 is known to be involved in the DNA damage response, we investigated the effect of its combination with Melphalan on HMCLs. Results show that this particular drug combination strongly enhances double strand breaks in HMCLs monitored using 53BP1 foci formation and gH2AX detection. This result emphasizes a potential role of SETD8 in DNA repair in MM cells. Furthermore, GSEA analysis of patients with high SETD8 expression highlighted a significant enrichment of genes involved in DNA repair, MYC-MAX targets and MAPK pathway. Our study is the first to demonstrate the importance of SETD8 for MM cells survival and suggest that SETD8 inhibition represent a promising strategy to improve conventional treatment of MM with DNA damaging agents. Disclosures No relevant conflicts of interest to declare.

TiO2nanoparticles induce DNA double strand breaks and cell cycle arrest in human alveolar cells

2014 ◽  
Vol 56 (2) ◽  
pp. 204-217 ◽  
Author(s):  
Krupa Kansara ◽  
Pal Patel ◽  
Darshini Shah ◽  
Ritesh K. Shukla ◽  
Sanjay Singh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Alveolar Cells ◽  
Strand Breaks ◽  
Cycle Arrest

scholarly journals De novo telomere addition at chromosome breaks: Dangerous Liaisons

2017 ◽  
Vol 216 (8) ◽  
pp. 2243-2245 ◽  
Author(s):  
Dmitri Churikov ◽  
Vincent Géli
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Dna Sequences ◽  
De Novo ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Telomeric Repeats ◽  
Strand Breaks ◽  
Chromosome Breaks ◽  
Cycle Dependent

Telomerase counteracts the loss of terminal DNA sequences from chromosome ends; however, it may erroneously add telomeric repeats to DNA double-strand breaks. In this issue, Ouenzar et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201610071) uncover cell cycle–dependent sequestration of the telomerase RNA in nucleoli, a process that excludes telomerase from DNA repair sites.

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