scholarly journals Cyclin D1 Promotes Survival and Chemoresistance By Maintaining ATR and CHEK1 Signaling in TP53-Deficient Mantle Cell Lymphoma Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5197-5197
Author(s):  
Suchismita Mohanty ◽  
Thai Tran ◽  
Natalie Sandoval ◽  
Atish Mohanty ◽  
Victoria Bedell ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Cell Cycle Checkpoints ◽  
Mantle Cell ◽  
Induced Apoptosis ◽  
Tp53 Status ◽  
Inducible Gene

Abstract Mantle cell lymphoma (MCL) is a heterogeneous disease, ranging from indolent to aggressive conditions. Prognostic markers that predict aggressive MCL include blastoid cytologic features, high proliferation index (Argatoff et al. 1997), INK4A/ARF locus deletion (Dreyling et al. 1997), TP53 deletion and/or mutations (Greiner et al. 1996), elevated cyclin D1 (CCND1) expression (Rosenwald et al. 2003), and NOTCH1/2 mutations (Kridel et al. 2012, Bea et al. 2013). Among these, TP53 lesions are the most recurrent, suggesting their important role in MCL pathogenesis. In response to DNA damage, TP53 in normal cells activates cell cycle checkpoints to stall DNA replication allowing time for DNA repair or induces apoptosis when damage is severe (Zhou and Elledge. 2000). Tumor cells lacking TP53 function rely on the ATR-CHEK1 signaling for cell cycle checkpoints following DNA damage (Powell et al. 1995). Although both TP53 deficiencies and elevated CCND1 expression levels have been associated with poor survival, possible cooperation of TP53 status and CCND1 expression in aggressive MCL has not been examined. In this study, we hypothesize that CCND1 overexpression collaborates with TP53 deficiency to promote MCL survival and chemoresistance. We compared the effects of CCND1 knockdown on cell survival and resistance to hydroxyurea (HU) and cytarabine to that of knockdown or pharmacological inhibition of CDK4 in MCL lines differing in TP53 status. Inducible gene knockdown was generated in UPN-1 cells to investigate the role of CCND1 in preventing replication stress and DNA damage and in the maintenance of the ATR and CHEK1 signaling. In addition, knockdown of TP53 in TP53-proficient MCL cells was performed to determine the contribution of TP53 status to tumor response to HU and the requirement of CCND1 in the chemosensitivity of these cells. We demonstrate that the survival of TP53-deficient MCL lines (UPN-1 and JEKO-1) is more dependent on CCND1 than on CDK4, but neither of these proteins is essential in TP53-proficient lines (REC-1 and Z-138). Using inducible gene knockdown in UPN-1 cells, we show that CCND1 depletion-induced apoptosis is caused by endogenous replication stress and DNA damage, which are related to defects in the DNA replication checkpoints ATR and CHEK1. The protective effect of CCND1 in MCL cell lines was also confirmed in vivo tumor model. Silencing of CCND1, but not CDK4, sensitizes TP53-deficient MCL cells to hydroxyurea (HU) or cytarabine, which activates the S-phase checkpoint. In addition, forced expression of CCND1 rescues TP53-deficient cells from HU-induced apoptosis in an ATR-dependent manner. In contrast, neither silencing of CCND1 nor CDK4 increases the sensitivity of TP53-proficient cells to these agents. Finally, knockdown of TP53 sensitizes REC-1 cells (TP53 competent) to combination of HU exposure and CCND1 inhibition, confirming the role of TP53 status and CCND1 expression in the chemosensitivity of MCL cells. In summary, these results uncover a novel role for CCND1 in maintaining the ATR and CHEK1 signaling in TP53-deficient MCL. This role of CCND1 could contribute to its oncogenic potential and chemoresistance in aggressive MCL that lack TP53. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Role of UBR5 Mutations in the Pathogenesis of Mantle CELL Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4124-4124
Author(s):  
Olga Kutovaya ◽  
Stacy Hung ◽  
Hughes Christopher ◽  
Randy D Gascoyne ◽  
Morin Gregg ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Cycle ◽  
Dna Damage ◽  
Mantle Cell Lymphoma ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
Interacting Proteins ◽  
Mantle Cell ◽  
Damage Response

Abstract Intro: Mantle cell lymphoma (MCL) accounts for 6% of non-Hodgkin lymphomas and represents a particularly challenging disease with patient outcomes inferior to most other lymphoma subtypes. Using targeted capture sequencing of MCL biopsy samples, we recently reported frequent mutations (18%) in UBR5, a gene encoding an E3 ubiquitin-protein ligase that has not been previously implicated in lymphomagenesis. All mutations were clustered within 100bp in or around exon 58 of UBR5 and truncate the reading frame or change a key lysine residue. These mutations are predicted to result in the loss of the conserved cysteine residue in the HECT-domain, which is responsible for binding the ubiquitin co-factor. The recurrence and clustering of UBR5 mutations suggest their critical pathogenic involvement in a subgroup of MCL that might be therapeutically targetable. The aim of this study is to determine UBR5 mutation-associated proteome changes and altered cell signaling. Methods: As seen in MCL patients, mutations in exon 58 of UBR5 were introduced to three MCL cell lines (Granta-519, Jeko-1, and Mino) using the CRISPR-Cas9 genome engineering tool. First, mass spectrometry-based immunoprecipitation proteomics (IP-MS) was employed to identify differences in UBR5 interacting partners between UBR5 mutant and wildtype (WT) cells. Candidate UBR5 interacting proteins were validated by flow cytometry, western blotting, co-immunoprecipitation, and immunofluorescence. Next, global proteomes of UBR5 mutants and WT were analyzed by Tandem Mass Tag (TMT)-based mass spectrometry quantification to identify proteins with differential expression due to the UBR5 mutations. Results: The IP-MS analysis of WT vs UBR5 mutants revealed histone and cell cycle control proteins as candidate differential UBR5 interacting proteins (p<0.05). Particularly, histones H1, H4, and H2AFX, as well as the cell cycle genes CDC5L, BUB3, MAP4, RAD50 and CDK11B were identified as candidate UBR5 interacting partners. The global proteome analysis identified a set of differentially expressed genes (mutant vs wt; p<0.05) that are common among the MCL cell lines with the same direction of change. Gene ontology analysis of this set revealed DNA damage response, chromosome organization, and cell cycle response pathways as the predominant pathways affected. Moreover, our preliminary functional studies indicate constitutive phosphorylation of H2AFX in UBR5 mutants vs WT in line with the role of UBR5 in DNA damage response. Conclusions: Our results are consistent with UBR5 functioning as a key regulator of cell signalling and strongly suggest UBR5 as a novel regulator of histone modifications and DNA damage response. These findings provide an experimentally valid platform for further functional investigation and testing of target therapies for MCL harbouring UBR5 mutations. Disclosures No relevant conflicts of interest to declare.

Pathogenesis of Mantle-Cell Lymphoma: All Oncogenic Roads Lead to Dysregulation of Cell Cycle and DNA Damage Response Pathways

2005 ◽  
Vol 23 (26) ◽  
pp. 6364-6369 ◽  
Author(s):  
Veronica Fernàndez ◽  
Elena Hartmann ◽  
German Ott ◽  
Elias Campo ◽  
Andreas Rosenwald
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
Mantle Cell ◽  
Damage Response ◽  
Cell Cycle Machinery

Mantle-cell lymphoma (MCL) is a well-defined subtype of B-cell non-Hodgkin's lymphomas (B-NHL), accounts for approximately 6% of all lymphoid neoplasms, and has a median survival of 3 to 4 years. The genetic hallmark of MCL is the chromosomal translocation t(11;14)(q13;q32) that leads to deregulation and upregulation of Cyclin D1, an important regulator of the G1 phase of the cell cycle. This genetic event is present in virtually all cases of MCL, whereas additional genetic alterations that occur in subsets of MCL have been described. Most of these alterations appear to disturb the cell cycle machinery/interfere with the cellular response to DNA damage, thus making MCL a paradigm for cell cycle and DNA damage response dysregulation in cancer in general. In particular, Cyclin D1 upregulation, genomic amplification of the cyclin-dependent kinase (CDK) -4, deletions of the CDK inhibitor p16INK4a and overexpression of BMI-1, a transcriptional repressor of the p16INK4a locus, are associated with dysregulation of the cell cycle machinery in MCL. The DNA damage response pathway is affected by frequent alterations of the ataxia-telangiectasia mutated (ATM) kinase as well as occasional inactivation of checkpoint kinase (CHK)-1 and CHK2 that are kinases that act downstream of ATM in response to detection of DNA damage. Moreover, p53 is frequently targeted by alterations in MCL. A recent gene expression profiling study defined the proliferation signature, a quantitative measure of gene expression of proliferation-associated genes as the strongest survival predictor available to date allowing the definition of prognostic MCL subgroups that differ in median survival by more than 5 years.

A Synthetic Lethal RNA Interference Screen Identifies Components of Genotoxic Stress Response As Targets for Anti-CCND1 Therapy in Mantle Cell Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2430-2430
Author(s):  
Suchismita Mohanty ◽  
Natalie Sandoval ◽  
Charles Warden ◽  
Vu N Ngo
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mantle Cell Lymphoma ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
Genotoxic Stress ◽  
Rnai Screen ◽  
Synthetic Lethal ◽  
Mantle Cell ◽  
Damage Response

Abstract Abstract 2430 Background. Almost all cases of mantle cell lymphoma (MCL) harbor the t(11:14) chromosomal translocation resulting in overexpression of the cell cycle regulatory protein cyclin D1 (CCND1), which promotes cell proliferation and poor survival. Targeting CCND1 in vitro and clinically, however, is not sufficient to cause tumor cell death, suggesting that additional mechanisms compensate for MCL growth and survival. Unraveling these additional signals will help identify novel targets for rational combination therapies in MCL. Previously, we developed a novel functional genomics tool using an inducible RNA interference (RNAi) library, which can simultaneously assess the role of thousands of genes in cell viability in tumor cell lines. Here we apply the inducible RNAi screen to identify synthetic lethal interactions with CCND1 in MCL. The screen uncovered several components of the DNA damage response as potential new combination targets for anti-CCND1 therapy in mantle cell lymphoma. Methods. The RNAi library was previously constructed using a retroviral vector that inducibly expresses small-hairpin RNA (shRNA). Each shRNA vector contains a unique 60-mer bar code that can hybridize to a corresponding complementary sequence spotted on a custom Agilent oligonucleotide microarray. To perform a synthetic lethal RNAi screen for CCND1, we first established a stable MCL line (UPN-1) that express an inducible CCND1 or control shRNA, transduced these lines with the pooled shRNA library, selected for transduced cells with puromycin, and induced shRNA expression for eight days. Genomic DNA containing bar code sequences was then amplified by PCR, fluorescently labeled, and hybridized onto microarrays. Each screen was repeated four times to enable statistical analysis. Candidate shRNAs obtained from the screen were validated for synergistic killing of MCL cells when combined with CCND1 knockdown. We evaluated genotoxic stress response triggered by DNA damage following CCND1 inactivation in MCL lines by Western blots. DNA damage and repair were assessed by comet assays and immuno-fluorescent staining of DNA repair proteins including phospho-H2AX, RAD51 and 53BP1. Results. The RNAi screen uncovers multiple shRNAs targeting RIPK1, RIPK3, NEMO, and TAK1, which sensitize MCL cells to CCND1 inhibition. RIPK1, NEMO, and TAK1 have been shown to play an essential role in cells undergoing genotoxic stress by linking DNA damage-induced ATM activation and NF-kB activity. We demonstrated that silencing CCND1 in the MCL cell lines UPN-1, JEKO-1, Z138, and Granta-519 up-regulates RIPK1 mRNA and protein expression, in addition to increased phosphorylation of DNA damage response proteins such as ATM, CHEK1/2 and H2AX. We observed a two-fold increase of DNA damage levels in CCND1 knockdown cells as assessed by comet assays. We also detected cell cycle-independent increase of DNA double strand break (DSB) foci in CCND1 knockdown cells by staining with fluorescently labeled anti-phospho-H2AX antibody. Knockdown of RIPK1 in MCL lines (UPN-1 and JEKO-1) resulted in apoptotic cell death and these RIPK1 shRNA-transduced cells are hypersensitive to irradiation and DNA damaging agents, indicating RIPK1 plays a protective role against DNA damage-induced apoptosis. The survival role of RIPK1 in MCL cells may correlate with the DNA repair function as demonstrated by the inability of RIPK1 knockdown cells to efficiently resolve etoposide-induced DNA DSB foci over time. Furthermore, we also found that RIPK1 knockdown cells failed to down-regulate the G2/M cell cycle checkpoint protein CDC25B and to up-regulate ATM phosphorylation and Ku86 protein expression in response to genotoxic stress. Blocking these RIPK1-dependent responses could sensitize MCL cells to CCND1 knockdown-induced DNA damage. Similar analyses of the other hits from the RNAi screen are on going. Conclusions. There are few viable treatment options for mantle cell lymphoma. We have identified the receptor interacting protein kinase 1 (RIPK1), of the DNA damage response pathway, as a potential therapeutic target whose downregulation sensitizes MCL cells to anti-CCND1 treatment, possibly by promoting insurmountable genotoxic stress. Successful implementation of our functional genetic screens for genes that sensitize MCL cells to anti-CCND1 treatment could define novel targets suitable for effective combination therapies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of UBR5 mutations in DNA damage response in mantle cell lymphoma

2017 ◽  
Vol 28 ◽  
pp. v583
Author(s):  
O. Kutovaya ◽  
S. Hung ◽  
E. Vigano ◽  
R. Gascoyne ◽  
G.B. Morin ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mantle Cell Lymphoma ◽  
Dna Damage Response ◽  
Cell Lymphoma ◽  
Mantle Cell ◽  
Damage Response

scholarly journals Activation of MYC, a bona fide client of HSP90, contributes to intrinsic ibrutinib resistance in mantle cell lymphoma

2018 ◽  
Vol 2 (16) ◽  
pp. 2039-2051 ◽  
Author(s):  
Jimmy Lee ◽  
Liang Leo Zhang ◽  
Wenjun Wu ◽  
Hui Guo ◽  
Yan Li ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Resistant Cell ◽  
Intrinsic Resistance ◽  
Mantle Cell ◽  
Myc Gene ◽  
Bona Fide ◽  
Ibrutinib Resistance ◽  
Induced Apoptosis

Abstract The BTK inhibitor ibrutinib has demonstrated a remarkable therapeutic effect in mantle cell lymphoma (MCL). However, approximately one-third of patients do not respond to the drug initially. To identify the mechanisms underlying primary ibrutinib resistance in MCL, we analyzed the transcriptome changes in ibrutinib-sensitive and ibrutinib-resistant cell lines on ibrutinib treatment. We found that MYC gene signature was suppressed by ibrutinib in sensitive but not resistant cell lines. We demonstrated that MYC gene was structurally abnormal and MYC protein was overexpressed in MCL cells. Further, MYC knockdown with RNA interference inhibited cell growth in ibrutinib-sensitive as well as ibrutinib-resistant cells. We explored the possibility of inhibiting MYC through HSP90 inhibition. The chaperon protein is overexpressed in both cell lines and primary MCL cells from the patients. We demonstrated that MYC is a bona fide client of HSP90 in the context of MCL by both immunoprecipitation and chemical precipitation. Furthermore, inhibition of HSP90 using PU-H71 induced apoptosis and caused cell cycle arrest. PU-H71 also demonstrates strong and relatively specific inhibition of the MYC transcriptional program compared with other oncogenic pathways. In a MCL patient-derived xenograft model, the HSP90 inhibitor retards tumor growth and prolongs survival. Last, we showed that PU-H71 induced apoptosis and downregulated MYC protein in MCL cells derived from patients who were clinically resistant to ibrutinib. In conclusion, MYC activity underlies intrinsic resistance to ibrutinib in MCL. As a client protein of HSP90, MYC can be inhibited via PU-H71 to overcome primary ibrutinib resistance.

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