Tumor Suppressor Microrna-29a/b and Microrna-34a Mediate Small Molecule PRIMA-1Met-Induced Aoptosis In Multiple Myeloma Cells By Targeting c-Myc

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1919-1919
Author(s):  
Manujendra N. Saha ◽  
Yijun Yang ◽  
Hong Chang
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Mutant P53 ◽  
Pcr Array ◽  
Wild Type ◽  
Down Regulation ◽  
Over Expression ◽  
P53 Status ◽  
Low Expression

Abstract PRIMA-1Met/APR246 (p53 reactivation and induction of massive apoptosis), is a small molecule with remarkable anti-tumor activities in various human tumor cells, and is currently under phase I/II clinical trial. We have previously demonstrated anti-tumor activity of PRIMA-1Met in multiple myeloma (MM) cells irrespective of p53 status. In addition, we have shown that PRIMA-1Met alone or in combination with dexamethasone triggers significant tumor growth inhibition in vivo in a murine xenograft model of human MM. However, the molecular mechanism underlying anti-myeloma activity of PRIMA-1Met has not been fully elucidated. MicroRNAs (miRNAs) are non-coding small RNA molecules that regulate post-transcriptional gene expression and play a critical role in tumor pathogenesis. Since the role of miRNAs and their regulation in response to PRIMA-1Met in MM is not known, here we investigated the relationship between PRIMA-1Met-induced apoptosis and miRNA expression in MM cells. Using a miRNA PCR array platform (Human Cancer Pathway Finder miScript miRNA PCR array, MIHS-102Z, Qiagen Inc), we analyzed the miRNA profiles in two MM cell lines of different p53 status (MM.1S with wild type p53 and 8226 with mutant p53) treated with either PRIMA-1Met or DMSO control. After normalization to a set of housekeeping genes, differential expressions of the miRNAs were analysed. miRNA-29a, miRNA-29b, and miRNA-34a were found significantly up-regulated (more than 2 fold, p<0.05) in cells treated with PRIMA-1Met compared to DMSO-treated cells. To evaluate the effect of over-expression of these miRNAs, we transfected two MM cell lines (MM.1S and 8226) with either miR-29a/b or miR-34a. Cells transfected with scramble miRNA were used as control. Over-expression of the miRNAs resulted in a dose-dependent inhibition of viability and increase in apoptosis of MM.1S or 8226 cells. Next, we examined the endogenous expression of these miRNAs in 5 primary MM samples by qPCR. Results showed a significant low expression of miR-29a/b and miR-34a in 3 of the 5 samples. Treatment of the two primary MM samples with low expression for miR-29a/b and miR-34a with PRIMA-1Met resulted in up-regulation of these miRNAs leading to inhibition of the viability and induction of apoptosis. To identify the possible targets of these miRNAs, we performed bioinformatics analysis. Results obtained from different searches by miRanda and TargetScan algorithm predicted c-Myc as a potential target for miRNA-29a/b and miRNA-34a. c-Myc is an oncogene whose over-expression has been associated with resistance to current chemotherapy in MM. Global gene expression profiling by microarray showed significant down-regulation of c-Myc in two MM cell lines with either wild type or mutant p53 treated with PRIMA-1Met compare to cells treated with DMSO. Importantly, down-regulation of c-Myc (∼2.6-fold) by PRIMA-1Met was also observed in a MM cell line (8226R5) lacking p53 expression suggesting an important role of c-Myc in p53-independent apoptosis of MM cells induced by PRIMA-1Met. By qPCR and Western blot analysis, we confirmed significant down-regulation of c-Myc in PRIMA-1Met-treated MM cells. These data provided the evidence for an inverse correlation between the expression of these miRNAs and c-Myc indicating that apoptosis of MM cells induced by PRIMA-1Met is regulated by miRNAs29a/b or miRNA34a targeting c-Myc. Our results suggest a novel mechanism for PRIMA-1Met-induced apoptotic signaling in MM cells mediated by up-regulation of miR-29a/b and miR-34a targeting c-Myc. Our findings also provide a preclinical framework for development of therapeutic strategies in combination of PRIMA-1Met and miRNA (miR-29a/b or miR-34a) mimics for the treatment of MM patients, especially for those with high c-Myc expressions. Disclosures: No relevant conflicts of interest to declare.

Cereblon Binding IMiD Small Molecules Mediate Myeloma Cell Death Via IRF4

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1807-1807
Author(s):  
Yuan Xiao Zhu ◽  
Chang-Xin Shi ◽  
Laura Bruins ◽  
Klaus Martin Kortuem ◽  
Jessica Schmidt ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Binding Site ◽  
Cell Lines ◽  
Protein Function ◽  
Research Funding ◽  
Wild Type ◽  
Binding Region ◽  
Down Regulation ◽  
Over Expression

Abstract Abstract 1807 We have recently demonstrated that cereblon (CRBN) mediates the direct anti-myeloma activity of immunomodulatory drugs (IMiDs). However, the genes/pathways downstream of CRBN associated with anti-myeloma activity remain unclear. We, and others, identified interferon regulatory factor 4 (IRF4) as one of the downstream targets of CRBN-associated signaling. Both lenalidomide treatment and CRBN knockdown downregulate IRF4. IRF4 levels return to baseline in IMiD resistant cells surviving CRBN silencing. To determine whether IMiD-induced IRF4 downregulation is critical to anti-MM activity, we overexpressed IRF4 in two IMiD-sensitive human MM cell lines (HMCLs), KMS11 and MM1.S, followed by lenalidomide treatment. Lenalidomide-induced cytotoxicity was greatly impaired in both HMCLs overexpressing IRF4 compared with the control virus infected cells. Further analysis indicated that IRF4 over-expression does not completely prevent lenalidomide-induced growth arrest, but reduces cell death by 70% after lenalidomide treatment. Immunoblotting analysis of KMS11 cells indicated that IRF4 over-expression blocks lenalidomide-induced activation of caspase 8, reduces up-regulation of p21waf and increases CDK6 expression but does not significantly affect lenalidomide-induced MYC down-regulation. Although cereblon and IRF4 are broadly expressed in MM, baseline levels of expression are only weakly correlated (r=0.22) in primary MM patient gene expression analysis. Gene expression studies revealed statistical changes in 1,368 genes when comparing high versus low CRBN expression in primary myeloma samples. Interestingly genes associated with high CRBN expression included cyclin D2, SOCS3 and IL4 while genes associated with low cereblon expression included cyclin D1, FRZB and CD200. In order to understand how CRBN is connected with downstream anti-myeloma signaling, a structure-function study was performed to determine which CRBN domain is required for lenalidomide-induced IRF4 down-regulation and cytotoxicity. Lentiviral constructs expressing wild-type CRBN and a series of mutated CRBN were generated, including mutations at thalidomide binding site (Y384A/W386A), deletion of DDB1 binding region (ΔMid) and truncations at N-terminal and C-terminal. Lentiviruses from these constructs were used to infect IMiDs resistant HMCLs, OCI-MY5 and MM1.S res. Both of these cell lines have very low endogenous CRBN expression and they became sensitive to lenalidomide after introduction of wild-type CRBN. Conversely, introduction of CRBN with mutated thalidomide-binding site or with DDB1 binding region depletion failed to mediate lenalidomide toxicity and down-regulation of IRF4. OCI-MY5 cells expressing either N-terminal or C-terminal truncated CRBN showed substantial reduced responses (more than 50%) to lenalidomide compared with wild-type CRBN expressing cells. Deletion of only 20–30 amino acids at either ends of CRBN greatly impaired the protein function, suggesting that protein folding might be important for CRBN-mediated IMiD response. Our data indicate that IMiD induced myeloma cytotoxicity is largely mediated by modifying CRBN associated E3 ubiqutin ligase and subsequent IRF4 downregulation, suggesting the CRBN-IRF4 axis is a potential target for development of new anti-myeloma drugs. Disclosures: Schmidt: Karyopharm: Research Funding. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Recurrent Mutations of Multiple Components of Cohesin Complex in Myeloid Neoplasms

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 782-782
Author(s):  
Ayana Kon ◽  
Lee-Yung Shih ◽  
Masashi Minamino ◽  
Masashi Sanada ◽  
Yuichi Shiraishi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Gene Mutations ◽  
Cohesin Complex ◽  
Wild Type ◽  
Myeloid Neoplasms ◽  
Whole Exome ◽  
Recurrent Mutations ◽  
Equity Ownership

Abstract Abstract 782 Recent genetic studies have revealed a number of novel gene mutations in myeloid malignancies, unmasking an unexpected role of deregulated histone modification and DNA methylation in both acute and chronic myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. In the previous study, we analyzed 29 paired tumor-normal samples with chronic myeloid neoplasms with myelodysplastic features using whole exome sequencing (Yoshida et al., Nature 2011). Although the major discovery was frequent spliceosome mutations tightly associated with myelodysplasia phenotypes, hundreds of unreported gene mutations were also identified, among which we identified recurrent mutations involving STAG2, a core cohesin component, and also two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex conserved across species and is composed of four core subunits, i.e., SMC1, SMC3, RAD21 and STAG proteins, together with several regulatory proteins. Forming a ring-like structure, cohesin is engaged in cohesion of sister chromatids in mitosis, post-replicative DNA repair and regulation of gene expression. To investigate a possible role of cohesin mutations in myeloid leukemogenesis, an additional 534 primary specimens of various myeloid neoplasms was examined for mutations in a total of 9 components of the cohesin and related complexes, using high-throughput sequencing. Copy number alterations in cohesin loci were also interrogated by SNP arrays. In total, 58 mutations and 19 deletions were confirmed by Sanger sequencing in 73 out of 563 primary myeloid neoplasms (13%). Mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205) and CML (8/65), with much lower mutation frequencies in MPN (2/76), largely in a mutually exclusive manner. In MDS, mutations were more frequent in RCMD and RAEB (19.5%) but rare in RA, RARS, RCMD-RS and 5q- syndrome (3.4%). Cohesin mutations were significantly associated with poor prognosis in CMML, but not in MDS cases. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles was performed in 19 cases with cohesin mutations. Majority of the cohesin mutations (16/19) existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we investigated a possible impact of mutations on cohesin functions, where 17 myeloid leukemia cell lines with or without cohesin mutations were examined for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of one or more components of cohesin was substantially reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we examined the effect of forced expression of wild-type cohesin on cell proliferation of cohesin-defective cells. Introduction of the wild-type RAD21 and STAG2 suppressed the cell growth of RAD21- (Kasumi-1 and MOLM13) and STAG2-defective (MOLM13) cell lines, respectively, supporting a leukemogenic role of compromised cohesin functions. Less frequent mutations of cohesin components have been described in other cancers, where impaired cohesion and consequent aneuploidy were implicated in oncogenic action. However, 23 cohesin-mutated cases of our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Alternatively, a growing body of evidence suggests that cohesin regulate gene expression, arguing for the possibility that cohesin mutations might participate in leukemogenesis through deregulated gene expression. Of additional note, the number of non-silent mutations determined by our whole exome analysis was significantly higher in 6 cohesin-mutated cases compared to non-mutated cases. Since cohesin also participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, we report a new class of common genetic targets in myeloid malignancies, the cohesin complex. Our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Induction of mucin gene expression in human colonic cell lines by PMA is dependent on PKC-ε

1999 ◽  
Vol 277 (5) ◽  
pp. G1041-G1047 ◽  
Author(s):  
D.-H. Hong ◽  
G. Petrovics ◽  
W. B. Anderson ◽  
J. Forstner ◽  
G. Forstner
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Dominant Negative ◽  
Mrna Levels ◽  
Wild Type ◽  
Mucin Gene ◽  
Time Period ◽  
Mucin Expression ◽  
Mucin Genes

Treatment of HT-29 cells with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induces MUC2 expression. To investigate the role of PKC in regulating mucin genes in intestinal cells, we examined the regulation of MUC1, MUC2, MUC5AC, MUC5B, and MUC6 expression in two human mucin-producing colonic cell lines, T84 and HT29/A1. T84 and HT29/A1 cells (at 80–90% confluency) were exposed to 100 nM PMA for 0, 3, and 6 h. Twofold or greater increases in mRNA levels for MUC2 and MUC5AC were observed in both cell lines during this time period, whereas the levels of MUC1, MUC5B, and MUC6 mRNAs were only marginally affected. These results indicated that PKC differentially regulates mucin gene expression and that it may be responsible for altered mucin expression. Our previous results suggested that the Ca2+-independent PKC-ε isoform appeared to mediate PMA-regulated mucin exocytosis in these cell lines. To determine if PKC-ε was also involved in MUC2/MUC5AC gene induction, HT29/A1 cells were stably transfected with either a wild-type PKC-ε or a dominant-negative ATP-binding mutant of PKC-ε (PKC-ε K437R). Overexpression of the dominant-negative PKC-ε K437R blocked induction of both mucin genes, whereas PMA-induced mucin gene expression was not prevented by overexpression of wild-type PKC-ε. PMA-dependent MUC2 mucin secretion was also blocked in cells overexpressing the dominant-negative PKC-ε K437R. On the basis of these observations, PKC-ε appears to mediate the expression of two major gastrointestinal mucins in response to PMA as well as PMA-regulated mucin exocytosis.

The Role of Mdm2 In Inactivation of the P53 Pathway In APL

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5111-5111
Author(s):  
Julia Abramowitz ◽  
Tzahi Neuman ◽  
Yoav Smith ◽  
Rania Samman ◽  
Alex Gural ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Tp53 Gene ◽  
Wild Type ◽  
P53 Pathway ◽  
Mdm2 Protein ◽  
Over Expression ◽  
Normal Bone ◽  
P53 Inactivation

Abstract Abstract 5111 The pathway controlled by the p53 tumor-suppressor protein is altered in most, if not all, human cancers and the TP53 gene itself is mutated in half of all human tumors. However, mutations in the TP53 gene are rare in human hematological malignancies. This implies that p53 is inactivated by alternative mechanisms such as over expression of its negative regulators. p53 is negatively regulated by Mdm2 through ubiquitin-dependent degradation and by Mdmx through inhibition of transcriptional function. To date, there is no information on the role of Mdm2 and Mdmx in human Acute Promyeloytic Leukemia (APL). We investigated the involvement of these negative regulators of p53 in APL at the gene and protein expression levels. First, we directly sequenced TP53 in 21 APL samples. In all cases TP53 was found to be wild type as expected. We studied Mdm2 and Mdmx gene expression in bone marrow samples of 30 APL patients at diagnosis in comparison to 35 normal bone marrow samples. Quantitative Real-Time PCR analysis showed no statistically significant differences in expression of Mdm2, Mdmx full length or its splicing variant (Mdmx-S) between APL and control samples. Bioinformatics analysis of gene expression of 39 APL patients at diagnosis, using publicly available arrays, showed homogeneous gene expression pattern between patients. We compared the APL arrays to 5 normal promyelocytes arrays. In agreement with our results, no significant difference was detected between APL and normal promyelocytes in the levels of Mdm2 or Mdmx gene expression. Mdm2 and Mdmx are subjected to complex regulation at the protein level. We therefore investigated the level of these proteins by immunohistochemical staining. Bone marrow biopsies from 23 APL patients at diagnosis were compared to 30 normal biopsies and protein levels were evaluated by semi-quantitative score. We found that Mdmx protein was low in APL samples and not significantly different from normal bone marrow. Thus, in APL, Mdmx does not appear to play a major role in p53 inhibition. Remarkably, APL samples showed a bi-modal expression of Mdm2 protein: 11/23 (48%) APL samples had significantly down-regulated Mdm2 protein (p<0.001). This might imply that Mdm2 is not involved in p53 inactivation in these patients. The other 12/23 (52%) patients showed significant over-expression of the Mdm2 protein (p<0.001). This result suggests that in half of APL patients, the p53 pathway may be inactivated by Mdm2. Interestingly, the subset of patients with high Mdm2 protein expression, was also characterized by high levels of p53 (5/12, p=0.058) and the pro-apoptotic p53 target, Puma (5/12). In summary, TP53 is wild type in APL. We found that the Mdm2 protein, a negative regulator of p53, may be accountable for p53 inactivation in about half of APL patients. In the other cases, a different, yet to be determined, mechanism is involved in inhibiting p53. Thus, APL is not a homogeneous disease regarding the inhibition of p53 pathway. Disclosures: No relevant conflicts of interest to declare.

Evidence for Mutant p53 Gain-of-Function Effects in Normal Haemopoietic Cells and Myc-Driven Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3589-3589
Author(s):  
Brandon James Aubrey ◽  
Andreas Strasser ◽  
Gemma Kelly ◽  
Lin Tai ◽  
Marco Herold
Keyword(s):  
Cell Death ◽  
Human Cancer ◽  
P53 Protein ◽  
Dominant Negative ◽  
Mutant P53 ◽  
P53 Mutations ◽  
Wild Type ◽  
Over Expression ◽  
Wild Type P53

Abstract Deregulated c-MYC expression and mutations in p53 are among the most common changes detected in human cancer. It is now established that mutant p53 proteins confer a poor prognosis in human cancer through both loss of wild-type p53 activity as well as various proposed gain-of-function properties. The specific role of mutant p53 in MYC-driven tumorigenesis is not known. The Eμ-Myc mouse model carries a c-Myc transgene under the control of the immunoglobulin heavy chain gene enhancer (Eμ), recapitulating the chromosomal translocation underlying human Burkitt Lymphoma (BL). These mice develop aggressive pre-B or B cell lymphomas and ~20% of those tumours exhibit p53 mutations. We have shown that MYC-driven lymphomas are exquisitely dependent on the pro-survival BCL-2 family member MCL-1 such that loss of a single allele of Mcl-1 leads to dramatic tumour regression and prolonged animal survival. Interestingly, we found that this dependency on MCL-1 is reduced, but not completely ablated, by the presence of a p53 mutation. This suggests an important role for mutant p53 in the sustained survival of MYC-driven lymphomas. We are investigating the effects of five different mutant mouse p53 proteins (V170M, I192S, G280, R246Q, R270H) on tumour initiation, sustained growth and chemoresistance in the Eμ-Myc mouse model. We are further examining the effect of p53 mutations on MCL-1 dependence by using a floxed Mcl-1 gene and a tamoxifen-inducible Cre-recombinase in established Eμ-Myc lymphomas. Preliminary data suggest that both loss of wild-type p53 function as well as retroviral over-expression of mutant p53 can compensate for reduced levels of MCL-1 (loss of one Mcl-1 allele). The underlying mechanisms for this are under investigation. The role of mutant p53 in lymphoma cell survival has been further examined in Eμ-Myc lymphoma-derived cell lines. Enforced over-expression of mutant p53 in cell lines containing wild-type p53 impaired induction of apoptosis by Nutlin3A, an inhibitor of Mdm-2 (the major negative regulator of p53). Remarkably, Nutlin-3a-induced apoptosis was impaired although it caused substantial transcriptional induction of the p53 apoptosis effectors, Puma and Noxa. Importantly, different mutant p53 proteins conferred different levels of protection against cell death. The observed protection against cell death may be partly due to dominant-negative effects of mutant p53, however, it does not appear to be robust enough to account for the extent of cell survival. Furthermore, mutant p53 conferred resistance to docetaxol, which is thought to induce cell death through predominantly p53-independent mechanisms. These data suggest that mutant p53 can protect against both p53-dependent and p53-independent cell death processes. Conversely, transcriptional induction of Noxa and Puma implies that “p53-restoration therapy” may remain a feasible treatment strategy even in tumours that bear mutations in p53 and that the role of a dominant-negative effect for some mutant p53 proteins may be less important than previously considered, at least in lymphoma cells. We are also examining the effect of mutant p53 on lymphoma development utilizing a hematopoietic reconstitution model and retroviral over-expression of mutant p53 proteins. The different mutant p53 proteins investigated exhibited distinct effects during tumorigenesis. The R246Q mutant p53 protein markedly accelerated lymphoma development in the context of MYC over-expression. The R246Q mutant p53 protein demonstrated strong selection in p53-deficient (p53-/-) hematopoietic cells during reconstitution indicative of an advantageous activity in emergency hematopoiesis. Overall, these findings provide evidence for a positive oncogenic role of mutant p53 in hematopoietic cells that provides a particularly potent selective advantage in the context of MYC driven lymphoma development. Importantly, different p53 mutations exhibit different functional properties such that different p53 mutations are likely to be associated with distinct risk in human malignant disease. Disclosures No relevant conflicts of interest to declare.

Effective Targeting Of The P53/MDM2 Axis In Preclinical Models Of Infant MLL-Rearranged Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 71-71 ◽  
Author(s):  
Richard B Lock ◽  
Jennifer Richmond ◽  
Laura High ◽  
Hernan Carol ◽  
Kathryn Evans ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Wild Type ◽  
Induction Type ◽  
P53 Status

Abstract Introduction While the overall cure rate for the most common pediatric cancer, acute lymphoblastic leukemia (ALL) now approaches 90%, infants (<12 months) diagnosed with ALL harboring translocations in the mixed-lineage leukemia oncogene (infant MLL-ALL) experience shorter remission duration and a significantly reduced likelihood of survival (∼50%). Therefore, new treatments that can be incorporated into conventional chemotherapy regimens to extend patient remission and improve survival are urgently required. Mutations in the p53 tumor suppressor are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be of therapeutic benefit. Nutlin cis-imidazole molecules selectively inhibit p53-MDM2 binding, resulting in activation of the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenograft models. Methods In vitro cytotoxicity was assessed by mitochondrial metabolic activity assay (Alamar blue) following 48h drug exposures. P53 protein levels and subcellular distribution were assessed by immunoblotting. Patient-derived xenografts were established from infant MLL-ALL, B-cell precursor (BCP)-ALL, or T-lineage ALL (T-ALL) bone marrow or peripheral blood (PB) biopsies in immune-deficient (NOD/SCID or NSG) mice, and their gene expression profiles generated using Illumina Human Ref-12 Expression BeadChips. Engraftment and drug responses were assessed by enumeration of the proportion of human versus mouse CD45+ cells in the PB. Mice with established disease received vehicle, RG7112 (100 mg/kg daily x 5 p.o.), a combination of vincristine (0.15 mg/kg once i.p.) dexamethasone (5 mg/kg daily x 5 i.p.) and L-asparaginase (1,000 IU/kg daily x 5 i.p.) (VXL), or RG7112 plus VXL. Anti-leukemic efficacy was assessed using an objective response measure modeled after the clinical setting, as well as the median event-free survival (EFS) of treated or control groups from treatment initiation. Therapeutic enhancement was considered to occur when the RG7112/VXL combination significantly extended mouse EFS compared with that of both of the RG7112 and VXL treated groups. Results Unsupervised hierarchical clustering of gene expression profiles revealed that the MLL-ALL (n=9), BCP-ALL (n=7) and T-ALL (n=13) xenografts clustered according to leukemia subtype. Moreover, genes previously reported to be overexpressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA gene family, were significantly upregulated in MLL-ALL xenografts. The specificity of RG7112 was validated by cytotoxicity assays against leukemia cell lines of known p53 status; p53 wild-type cell lines (RS4;11, IC50 1.4 µM; NALM-6, IC50 3.0 µM) were markedly more sensitive than those with mutant p53 (CEM, IC50 >10 µM; JURKAT, IC50 >10 µM). The in vitro sensitivity of BCP-ALL (n=3) and infant MLL-ALL (n=4) xenografts was consistent with wild-type p53 status, with IC50s of 0.11 - 2.2 µM. Exposure of ALL xenograft cells to nutlin-3 (10 µM, 6h) caused marked p53 up-regulation and nuclear translocation. Since we had previously shown that RG7112 administered as a single agent for 14 days induced significant regressions [Complete Responses (CRs) or greater] in 7/7 infant MLL-ALL xenografts in vivo, we assessed its efficacy in a 5-day combination treatment with an induction-type regimen (VXL) against two infant MLL-ALL xenografts (MLL-5 and MLL-14). The RG7112/VXL combination caused a Partial Response in MLL-5 compared with Progressive Disease for both RG7112 and VXL. The efficacy of RG7112/VXL was even more pronounced against MLL-14, causing a Maintained CR compared with CRs for both RG7112 and VXL, which met the criteria for Therapeutic Enhancement (the median EFS of RG7112/VXL-treated mice, 65.0 days, was significantly greater, P< 0.0001, than that of RG7112, 22.2 days, and VXL, 28.5 days). Conclusions RG7112 induces significant regressions in a high proportion of infant MLL-ALL xenografts and enhances the efficacy of an induction-type regimen. The utility of targeting the p53-MDM2 axis in combination with established drugs for the clinical management of infant MLL-ALL warrants further investigation. This study was supported by NCI NO1CM42216. The authors thank Roche Pharmaceuticals, Inc., for providing RG7112. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Rereplication by Depletion of Geminin Is Seen Regardless of p53 Status and Activates a G2/M Checkpoint

2004 ◽  
Vol 24 (16) ◽  
pp. 7140-7150 ◽  
Author(s):  
Wenge Zhu ◽  
Yuefeng Chen ◽  
Anindya Dutta
Keyword(s):  
Dna Replication ◽  
Human Cancer ◽  
Genomic Stability ◽  
Mutant P53 ◽  
Wild Type ◽  
Human Cancer Cells ◽  
P53 Status ◽  
Giant Nuclei ◽  
Cdc2 Activity

ABSTRACT Genomic DNA replication is tightly controlled to ensure that DNA replication occurs once per cell cycle; loss of this control leads to genomic instability. Geminin, a DNA replication inhibitor, plays an important role in regulation of DNA replication. To investigate the role of human geminin in the maintenance of genomic stability, we eliminated geminin by RNA interference in human cancer cells. Depletion of geminin led to overreplication and the formation of giant nuclei in cells that had wild-type or mutant p53. We found that overreplication caused by depletion of geminin activated both Chk1 and Chk2, which then phosphorylated Cdc25C on Ser216, resulting in its sequestration outside the nucleus, thus inhibiting cyclin B-Cdc2 activity. This activated G2/M checkpoint prevented cells with overreplicated DNA from entering mitosis. Addition of caffeine, UCN-01, or inhibitors of checkpoint pathways or silencing of Chk1 suppressed the accumulation of overreplicated cells and promoted apoptosis. From these results, we conclude that geminin is required for suppressing overreplication in human cells and that a G2/M checkpoint restricts the proliferation of cells with overreplicated DNA.

scholarly journals PRIMA-1met Induces Autophagy in Colorectal Cancer Cells With Different p53 Status

2020 ◽  
Author(s):  
Xiao-lan Li ◽  
Jianbiao Zhou ◽  
Chen-jing Xia ◽  
Zhong-kai Lu ◽  
Zhi-rong Chen
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Cell Counting ◽  
Signaling Cascade ◽  
Mutant P53 ◽  
Wild Type ◽  
P53 Status ◽  
Wild Type P53 ◽  
In Cells

Abstract Background: PRIMA-1met (APR246), a methylated form of PRIMA-1 (p53-reactivation and induction of massive apoptosis-1, APR-017), targets mutant p53 for restoring its wild-type structure and function. We previously demonstrated that PRIMA-1met was efficient in suppressing the growth of colorectal cancer (CRC) cells in a p53-independent manner, and distinctly induced apoptosis mediated by up-regulation of Noxa in p53-mutant cell lines. Here we aimed to the effect of PRIMA-1met on autophagy in different CRC cell lines, to further investigate mechanisms underlying the inhibitory effect in cells with different p53 status.Methods: 3 CRC cell lines with wild-type p53, 5 lines with mutant p53 and 1 line without p53 were obtained for this study. Using western blotting, acridine orange staining, and transmission electron microscopy detection, we assessed autophagy flux in different cells treated with PRIMA-1met, and detected expression of mTOR/AMPK-ULK1-Vps34 autophagic signaling cascade. We also evaluated cell proliferation of cells with PRIMA-1met treatment by cell counting Kit-8 proliferation assay, compared to combination of PRIAM-1met and 3-Methyladenine. Furthermore, we knocked down Noxa gene by siRNA in different CRC cells, to assess LC3 conversion after administration of PRIMA-1met. Values were expressed as mean + standard error of the mean. Comparison between groups of data was made using one-way analysis of variance.Results: In this study, we showed that PRIMA-1met induced autophagy in CRC cells independent on p53 status. PRIMA-1met not only promoted autophagic vesicles (AVs) formation and AV-lysosome fusion, but also increased lysosomal degradation. Mechanistically, activation of mTOR/AMPK-ULK1-Vps34 autophagic signaling cascade was important for PRIMA-1met-induced autophagy. Furthermore, autophagy played a crucial role in the inhibitory effect of PRIMA-1met only in cells harboring wild-type p53, which was closely related to the increased Noxa.Conclusions: Our results indicated that PRIMA-1met induced autophagy in CRC cells regardless of p53 status via activating mTOR/AMPK-ULK1-Vps34 signaling cascade. However, induced autophagy was relevant to the cytotoxicity of PRIMA-1met in cells carrying wild-type p53, along with up-regulation of Noxa. Implying that, PRIMA-1met-based therapy could be an effective strategy for CRC.Trail registration: Not applicable.

scholarly journals The Anti-Tumor Activity of the NEDD8 Inhibitor Pevonedistat in Neuroblastoma

2021 ◽  
Vol 22 (12) ◽  
pp. 6565
Author(s):  
Jennifer H. Foster ◽  
Eveline Barbieri ◽  
Linna Zhang ◽  
Kathleen A. Scorsone ◽  
Myrthala Moreno-Smith ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Wild Type ◽  
Tumor Weight ◽  
Cycle Arrest ◽  
P53 Status ◽  
Neuroblastoma Cell Lines ◽  
Anti Tumor Activity

Pevonedistat is a neddylation inhibitor that blocks proteasomal degradation of cullin–RING ligase (CRL) proteins involved in the degradation of short-lived regulatory proteins, including those involved with cell-cycle regulation. We determined the sensitivity and mechanism of action of pevonedistat cytotoxicity in neuroblastoma. Pevonedistat cytotoxicity was assessed using cell viability assays and apoptosis. We examined mechanisms of action using flow cytometry, bromodeoxyuridine (BrDU) and immunoblots. Orthotopic mouse xenografts of human neuroblastoma were generated to assess in vivo anti-tumor activity. Neuroblastoma cell lines were very sensitive to pevonedistat (IC50 136–400 nM). The mechanism of pevonedistat cytotoxicity depended on p53 status. Neuroblastoma cells with mutant (p53MUT) or reduced levels of wild-type p53 (p53si-p53) underwent G2-M cell-cycle arrest with rereplication, whereas p53 wild-type (p53WT) cell lines underwent G0-G1 cell-cycle arrest and apoptosis. In orthotopic neuroblastoma models, pevonedistat decreased tumor weight independent of p53 status. Control mice had an average tumor weight of 1.6 mg + 0.8 mg versus 0.5 mg + 0.4 mg (p < 0.05) in mice treated with pevonedistat. The mechanism of action of pevonedistat in neuroblastoma cell lines in vitro appears p53 dependent. However, in vivo studies using mouse neuroblastoma orthotopic models showed a significant decrease in tumor weight following pevonedistat treatment independent of the p53 status. Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, deserve further study in the treatment of neuroblastoma.

scholarly journals The nucleoplasmic interactions among Lamin A/C-pRB-LAP2α-E2F1 are modulated by dexamethasone

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anastasia Ricci ◽  
Sara Orazi ◽  
Federica Biancucci ◽  
Mauro Magnani ◽  
Michele Menotta
Keyword(s):  
Gene Expression ◽  
Cell Cycle Progression ◽  
Regulation Of Gene Expression ◽  
Lamin A ◽  
Wild Type ◽  
Cycle Progression ◽  
C Dynamics ◽  
E2f Transcription Factor ◽  
Transcription Factor 1

AbstractAtaxia telangiectasia (AT) is a rare genetic neurodegenerative disease. To date, there is no available cure for the illness, but the use of glucocorticoids has been shown to alleviate the neurological symptoms associated with AT. While studying the effects of dexamethasone (dex) in AT fibroblasts, by chance we observed that the nucleoplasmic Lamin A/C was affected by the drug. In addition to the structural roles of A-type lamins, Lamin A/C has been shown to play a role in the regulation of gene expression and cell cycle progression, and alterations in the LMNA gene is cause of human diseases called laminopathies. Dex was found to improve the nucleoplasmic accumulation of soluble Lamin A/C and was capable of managing the large chromatin Lamin A/C scaffolds contained complex, thus regulating epigenetics in treated cells. In addition, dex modified the interactions of Lamin A/C with its direct partners lamin associated polypeptide (LAP) 2a, Retinoblastoma 1 (pRB) and E2F Transcription Factor 1 (E2F1), regulating local gene expression dependent on E2F1. These effects were differentially observed in both AT and wild type (WT) cells. To our knowledge, this is the first reported evidence of the role of dex in Lamin A/C dynamics in AT cells, and may represent a new area of research regarding the effects of glucocorticoids on AT. Moreover, future investigations could also be extended to healthy subjects or to other pathologies such as laminopathies since glucocorticoids may have other important effects in these contexts as well.

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