nutlin-3a and Lymphoma

Nutlin3a is a small-molecule antagonist of MDM2 that promotes non-genotoxic activation of p53 through p53 protein stabilization and transactivation of p53 target genes. Nutlin3a is the forerunner of a class of cancer therapeutics that have reached clinical trials. Using transgenic and gene-targeted mouse models lacking the critical p53 target genes, p21, Puma, and Noxa, we found that only loss of PUMA conferred profound protection against Nutlin3a-induced killing in both non-transformed lymphoid cells and Eμ-Myc lymphomas in vitro and in vivo. CRISPR/Cas9-mediated targeting of the PUMA gene rendered human hematopoietic cancer cell lines markedly resistant to Nutlin3a-induced cell death. These results demonstrate that PUMA-mediated apoptosis, but not p21-mediated cell-cycle arrest or senescence, is a critical determinant of the therapeutic response to non-genotoxic p53 activation by Nutlin3a. Importantly, in human cancer, PUMA expression may predict patient responses to treatment with MDM2 antagonists.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Cell Cycle Checkpoints; Cell Line, Tumor; CRISPR-Cas Systems; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Lymphoma; Mice; Mice, Inbred C57BL; Mice, Transgenic; Piperazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The Epstein-Barr virus (EBV) is associated with various lymphoproliferative disorders and lymphomas. We have previously demonstrated that treating wild-type TP53-expressing B cell lines with the TP53 pathway activator nutlin-3 induced apoptosis in EBV-negative and EBV-positive latency I cells whereas EBV-positive latency III cells remained much more apoptosis-resistant. Here, we report a constitutively high level of autophagy in these resistant cells which express high levels of the proautophagic protein BECN1/Beclin 1 based, at least in part, on the activation of the NFKB signaling pathway by the viral protein LMP1. Following treatment with nutlin-3, several autophagy-stimulating genes were upregulated both in EBV-negative and EBV-positive latency III cells. However the process of autophagy was only triggered in the latter and was associated with an upregulation of SESN1/sestrin 1 and inhibition of MTOR more rapid than in EBV-negative cells. A treatment with chloroquine, an inhibitor of autophagy, potentiated the apoptotic effect of nutlin-3, particularly in those EBV-positive cells which were resistant to apoptosis induced by nutlin-3 alone, thereby showing that autophagy participates in this resistant phenotype. Finally, using immunohistochemical staining, clinical samples from various B cell lymphoproliferations with the EBV-positive latency II or III phenotype were found to harbor a constitutively active autophagy.

Topics: Apoptosis; Autophagy; B-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Gene Expression; Herpesvirus 4, Human; Humans; Imidazoles; Lymphoma; Piperazines; Tumor Suppressor Protein p53

The P53 tumor suppressor protein is a multifunctional transcription factor that prevents the malignant transformation of normal cells. In human malignancies, p53 is the most frequently altered gene and is mutated in approximately 50% of all malignancies. In contrast, p53 gene mutation has been rarely detected in feline malignancies, and most feline malignancies conceivably retain the wild-type p53 (wt-p53) gene. MDM2 negatively regulates the P53 protein by inhibiting its transcriptional activity and nuclear transport and by inducing its degradation. Inhibition of P53-MDM2 interaction stabilizes P53 protein and activates P53 pathway. Nutlin-3, a small molecule that inhibits P53-MDM2 interaction, was shown to have an antitumor effect in several human cancer cells retaining the wt-p53 gene. In the present study, we evaluated and compared the antitumor effect of nutlin-3 in 5 different feline lymphoma cell lines, of which 3 harbored wt-p53, and 2, mutated p53 (mt-p53). Treatment with nutlin-3 resulted in increased amounts of P53 protein in conjunction with augmented expression of P53-target genes in 3 feline lymphoma cell lines with the wt-p53 gene, but not in 2 feline lymphoma cell lines with the mt-p53 gene. Nutlin-3 treatment also induced G1-S and/or G2-M cell cycle arrest and apoptosis in lymphoma cell lines with wt-p53. Nutlin-3 treatment induced cell cycle arrest but not apoptosis in the cell lines with mt-p53. From these results, we concluded that nutlin-3 has an antitumor effect on feline lymphoma cell lines harboring the wt-p53 gene through accumulation and activation of P53 leading to cell cycle arrest and apoptosis. The present study suggests that inhibition of P53-MDM2 interaction using nutlin-3 may be a new therapeutic strategy for treating feline lymphoma retaining the wt-p53 gene.

Topics: Animals; Antineoplastic Agents; Apoptosis; Base Sequence; Cat Diseases; Cats; Cell Cycle Checkpoints; Cell Line, Tumor; DNA, Neoplasm; Genes, p53; Humans; Imidazoles; Lymphoma; Mutation; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

MicroRNAs (miRNAs) might behave as tumor suppressors and for that they are under consideration as novel therapeutic drugs. We tested the tumor suppressor activity of miRNA-34a (miR-34a) by measuring cell proliferation of the follicular lymphoma cell line DoHH2 transfected with this miRNA. We report that miR-34a did not inhibit cell proliferation notwithstanding a marked down-regulation of c-MYC. Interestingly, DoHH2 transfected cells showed a significant p53 down-regulation, suggesting that c-MYC positively controls p53 and the failed inhibition of cell proliferation is probably due to the down-regulation of the c-MYC/p53 axis. In keeping with this, c-MYC silencing also down-regulated p53 and had no effect on cell proliferation. In accordance with this hypothesis, etoposide or nutlin-3 treatment or a small interfering RNA (siRNA) against BCL6 (B-cell lymphoma 6) inhibited the proliferation of DoHH2 cells by up-regulating p53 without affecting either miR-34a or c-MYC levels. These results indicate that the proliferation is controlled by the regulatory axis c-MYC/p53 and suggest that paradoxically miR-34a behaves as a pro-proliferative rather than an anti-proliferative miRNA in DoHH2 cells.

Topics: 3' Untranslated Regions; Antineoplastic Agents, Phytogenic; Base Sequence; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Down-Regulation; Etoposide; Gene Expression; Green Fluorescent Proteins; Humans; Imidazoles; Lymphoma; MicroRNAs; Piperazines; Proto-Oncogene Proteins c-bcl-6; Proto-Oncogene Proteins c-myc; RNA Interference; Transfection; Tumor Suppressor Protein p53

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is associated with Kaposi's sarcoma (KS) as well as primary effusion lymphomas (PEL). The expression of viral proteins capable of inactivating the p53 tumor suppressor protein has been implicated in KSHV oncogenesis. However, DNA-damaging drugs such as doxorubicin are clinically efficacious against PEL and KS, suggesting that p53 signaling remains intact despite the presence of KSHV. To investigate the functionality of p53 in PEL, we examined the response of a large number of PEL cell lines to doxorubicin. Two out of seven (29%) PEL cell lines harbored a mutant p53 allele (BCBL-1 and BCP-1) which led to doxorubicin resistance. In contrast, all other PEL containing wild-type p53 showed DNA damage-induced cell cycle arrest, p53 phosphorylation, and p53 target gene activation. These data imply that p53-mediated DNA damage signaling was intact. Supporting this finding, chemical inhibition of p53 signaling in PEL led to doxorubicin resistance, and chemical activation of p53 by the Hdm2 antagonist Nutlin-3 led to unimpaired induction of p53 target genes as well as growth inhibition and apoptosis.

Topics: Animals; Cell Cycle Proteins; Cell Line; Cell Transformation, Viral; DNA Damage; Doxorubicin; Drug Resistance, Neoplasm; Gene Expression; Herpesviridae Infections; Herpesvirus 8, Human; Imidazoles; Lymphoma; Phosphorylation; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent for primary effusion lymphoma (PEL), a non-Hodgkin type lymphoma manifesting as an effusion malignancy in the affected individual. Although KSHV has been recognized as a tumor virus for over a decade, the pathways for its tumorigenic conversion are incompletely understood, which has greatly hampered the development of efficient therapies for KSHV-induced malignancies like PEL and Kaposi's sarcoma. There are no current therapies effective against the aggressive, KSHV-induced PEL. Here we demonstrate that activation of the p53 pathway using murine double minute 2 (MDM2) inhibitor Nutlin-3a conveyed specific and highly potent activation of PEL cell killing. Our results demonstrated that the KSHV latency-associated nuclear antigen (LANA) bound to both p53 and MDM2 and that the MDM2 inhibitor Nutlin-3a disrupted the p53-MDM2-LANA complex and selectively induced massive apoptosis in PEL cells. Together with our results indicating that KSHV-infection activated DNA damage signaling, these findings contribute to the specificity of the cytotoxic effects of Nutlin-3a in KSHV-infected cells. Moreover, we showed that Nutlin-3a had striking antitumor activity in vivo in a mouse xenograft model. Our results therefore present new options for exploiting reactivation of p53 as what we believe to be a novel and highly selective treatment modality for this virally induced lymphoma.

Topics: Animals; Cell Cycle; Cell Survival; DNA Damage; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Genes, p53; Herpesvirus 4, Human; Herpesvirus 8, Human; Humans; Imidazoles; Lymphoma; Mice; Piperazines; Sarcoma, Kaposi; Transplantation, Heterologous; Tumor Suppressor Protein p53; Virus Latency