nutlin-3a and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

Telomere erosion in cells with insufficient levels of the telomerase reverse transcriptase (TERT), contributes to age-associated tissue dysfunction and senescence, and p53 plays a crucial role in this response. We undertook a genome-wide CRISPR screen to identify gene deletions that sensitized p53-positive human cells to telomerase inhibition. We uncovered a previously unannotated gene, C16ORF72, which we term Telomere Attrition and p53 Response 1 (TAPR1), that exhibited a synthetic-sick relationship with TERT loss. A subsequent genome-wide CRISPR screen in TAPR1-disrupted cells reciprocally identified TERT as a sensitizing gene deletion. Cells lacking TAPR1 or TERT possessed elevated p53 levels and transcriptional signatures consistent with p53 upregulation. The elevated p53 response in TERT- or TAPR1-deficient cells was exacerbated by treatment with the MDM2 inhibitor and p53 stabilizer nutlin-3a and coincided with a further reduction in cell fitness. Importantly, the sensitivity to treatment with nutlin-3a in TERT- or TAPR1-deficient cells was rescued by loss of p53. These data suggest that TAPR1 buffers against the deleterious consequences of telomere erosion or DNA damage by constraining p53. These findings identify C16ORF72/TAPR1 as new regulator at the nexus of telomere integrity and p53 regulation.

Topics: Aminobenzoates; Cell Line, Tumor; DNA Damage; Gene Knockout Techniques; Humans; Imidazoles; Intercellular Signaling Peptides and Proteins; Naphthalenes; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Telomerase; Telomere; Transduction, Genetic; Tumor Suppressor Protein p53; Up-Regulation

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is triggered by BCR/ABL kinase. Recent efforts focused on the development of more potent tyrosine kinase inhibitors (TKIs) that also inhibit mutant tyrosine kinases such as nilotinib and dasatinib. Although major advances in the treatment of this aggressive disease with potent inhibitors of the BCR/ABL kinases, patients in remission frequently relapse due to drug resistance possibly mediated, at least in part, by compensatory activation of growth-signaling pathways and protective feedback signaling of leukemia cells in response to TKI treatment. Continuous activation of AKT/mTOR signaling and inactivation of p53 pathway were two mechanisms of TKI resistance. Here, we reported that nutlin-3 plus tanshinone IIA significantly potentiated the cytotoxic and apoptotic induction effects of imatinib by down-regulation of the AKT/mTOR pathway and reactivating the p53 pathway deeply in Ph+ ALL cell line. In primary samples from Ph+ ALL patients, nutlin-3 plus tanshinone IIA also exhibited synergetic cytotoxic effects with imatinib. Of note, three samples from Ph+ ALL patients harboring T315I mutation also showed sensitivity to the combined treatment of imatinib, nutlin-3 plus tanshinone IIA. In Ph+ ALL mouse models, imatinib combined with nutlin-3 plus tanshinone IIA also exhibited synergetic effects on reduction in leukemia burden. These results demonstrated that nutlin-3 plus tanshinone IIA combined TKI might be a promising treatment strategy for Ph+ ALL patients.

Topics: Abietanes; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Dose-Response Relationship, Drug; Drug Synergism; Female; Genes, p53; Humans; Imatinib Mesylate; Imidazoles; K562 Cells; Mice; Mice, Inbred NOD; Mice, SCID; Oncogene Protein v-akt; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Signal Transduction; TOR Serine-Threonine Kinases; Treatment Outcome

MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph-) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph- ALL.

Topics: Adult; Aged; Antineoplastic Agents; Cell Survival; Female; Humans; Imidazoles; Male; Middle Aged; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-mdm2; Tumor Cells, Cultured; Tumor Suppressor Protein p53

ETV6/RUNX1 (E/R) is the most common fusion gene in childhood acute lymphoblastic leukemia. It is responsible for the initiation of leukemia but also indispensable for disease maintenance and propagation, although its function in these latter processes is less clear. We therefore investigated the effects of the perceived p53 pathway alterations in model cell lines and primary leukemias and, in particular, how E/R upregulates MDM2, the predominant negative regulator of p53. We found that E/R transactivates MDM2 in both p53(+/+) and p53(-/-) HCT116 cells by binding to promoter-inherent RUNX1 motifs, which indicates that this activation occurs in a direct and p53-independent manner. Treatment of E/R-positive leukemic cell lines with Nutlin-3, a small molecule that inhibits the MDM2/p53 interaction, arrests their cell cycle and induces apoptosis. These phenomena concur with a p53-induced expression of p21, pro-apoptotic BAX and PUMA, as well as caspase 3 activation and poly ADP-ribose polymerase cleavage. The addition of DNA-damaging and p53-activating chemotherapeutic drugs intensifies apoptosis. Moreover, Nutlin-3 exposure leads to an analogous p53 accumulation and apoptotic surge in E/R-positive primary leukemic cells. Our findings clarify the role of p53 signaling in E/R-positive leukemias and outline the potential basis for its therapeutic exploitation in this setting.

Topics: Apoptosis; Child; Chromatin Immunoprecipitation; Core Binding Factor Alpha 2 Subunit; ETS Translocation Variant 6 Protein; Humans; Imidazoles; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-ets; Proto-Oncogene Proteins c-mdm2; Real-Time Polymerase Chain Reaction; Repressor Proteins; Signal Transduction; Transcription, Genetic; Tumor Suppressor Protein p53

In pediatric acute lymphoblastic leukemia (ALL), overexpression of murine double minute 2 (MDM2) protein by leukemic cells is typically associated with a wild-type (wt)-p53 phenotype and chemoresistance. A recently developed small-molecule antagonist of MDM2, nutlin-3, inhibits the MDM2-p53 interaction, resulting in induction of p53 activity and apoptosis. In this study, we evaluated the cytotoxic effect of nutlin-3 on ALL cells with different p53 status and MDM2 expression, using 18 cell lines and 30 primary leukemia samples. We found that both ALL cell lines and primary ALL samples with wt-p53 are sensitive to nutlin-3. No cytotoxic effect of nutlin-3 was detected in ALL cells with either p53-mutant or -null phenotype. In wt-p53 ALL cells, there was a significant positive correlation between MDM2 expression levels and sensitivity to nutlin-3. Nutlin-3-induced cell death was mediated by p53-induced activation of proapoptotic proteins and by p53-induced repression of the anti-apoptotic protein survivin. As p53 function is inhibited by MDM2 in chemoresistant, MDM2-overexpressing ALL cells, potent killing of these cells by nutlin-3 suggests that this agent may be a novel therapeutic for refractory ALL.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Child; Gene Expression; Humans; Imidazoles; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-mdm2; Tumor Cells, Cultured; Tumor Suppressor Protein p53