nutlin-3a and Leukemia

The small molecule Nutlin-3 is a potent antagonist of the murine double minute 2 (MDM2)/p53 interaction exhibiting promising therapeutic anti-cancer activity. Nutlin-3 has been proposed as an anti-neoplastic agent for the treatment of onco-hematological diseases characterized by a lower incidence of p53 mutation with respect to solid tumors. Indeed, based on its selective non-genotoxic p53 activation, Nutlin-3 might represent an alternative to the current cytotoxic chemotherapy. To overcome the poor bioavailability of Nutlin-3, we have assessed the potential efficacy of Nutlin-3 loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) against hematological malignancies. To test the specificity of the anti-leukemic activity, we have used leukemic cell lines characterized by different p53 status (JVM-2 and BJAB). NP loaded with Nutlin-3 (NP-Nutlin) were rapidly taken up by the leukemic cells and were as effective as native Nutlin-3 in promoting both induction of apoptosis and cell cycle arrest in p53(wild-type) JVM-2 cells, but not in p53(mutated) BJAB cells. Moreover, injection of NP-Nutlin, but not of free Nutlin-3, in a JVM-2-derived xenograft mouse model, reduced the subcutaneous tumor volume and promoted induction of apoptosis in the tumor mass. Overall, the chemical and structural characteristics of the NP-Nutlin-3, as well as their biological activity in vitro and in vivo, made them promising for further preclinical evaluations as potentially useful anti-leukemic carriers.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Humans; Imidazoles; Leukemia; Nanoparticles; Piperazines; Polyglactin 910; Tumor Suppressor Protein p53

P53 dysfunction has been associated with various malignant tumors, including acute leukemia. The overexpression of mouse double minute 2 (MDM2) causes the inactivation of p53 in acute leukemia. MDM2 inhibitors that activate p53 and induce apoptosis are currently being developed for potential treatment of acute leukemia. However, MDM2 inhibitors alone have limited efficacy in acute leukemia therapeutics. Combining other drugs to enhance the efficacy of MDM2 inhibitors is the thus considered as a potential treatment scheme. Here, we report that the combination of Nutlin-3 and Tanshinone IIA synergistically induces cytotoxicity, cell cycle arrest, apoptosis, and autophagic cell death, thereby imparting anti-leukemia effect in an acute leukemia cell line with wild-type p53 by effectively activating p53, inhibiting the AKT/mTOR pathway, and activating the RAF/MEK pathway. Using primary samples from acute leukemia patients, we show that the combination of Nutlin-3 plus Tanshinone IIA synergistically induces cytotoxicity by activating p53 and inhibiting the AKT/mTOR pathway. This specific combination of Nutlin-3 and Tanshinone IIA is also effective in preventing the recurrence of refractory leukemia, such as Ph+ ALL with the ABL kinase T315I mutation and AML with the FLT3-ITD mutation. Taken together, the results of this study demonstrate that the Nutlin-3 plus Tanshinone IIA combination exerts synergistic anti-leukemia effects by regulating the p53 and AKT/mTOR pathways, although further investigation is warranted. Small-molecule MDM2 antagonists plus Tanshinone IIA may thus be a promising strategy for the treatment of acute leukemia.

Topics: Acute Disease; Adolescent; Adult; Aged, 80 and over; Benzofurans; Cytotoxins; Drug Synergism; Female; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Imidazoles; Leukemia; Male; Middle Aged; Piperazines; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-mdm2; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

MDM2 (mouse double minute 2) inhibitors that activate p53 and induce apoptosis in a non-genotoxic manner are in clinical development for treatment of leukemias. P53 can modulate other programmed cell death pathways including autophagy both transcriptionally and non-transcriptionally. We investigated autophagy induction in acute leukemia by Nutlin 3a, a first-in-class MDM2 inhibitor. Nutlin 3a induced autophagy in a p53 dependent manner and transcriptional activation of AMP kinase (AMPK) is critical, as this effect is abrogated in AMPK -/- mouse embryonic fibroblasts. Nutlin 3a induced autophagy appears to be pro-apoptotic as pharmacological (bafilomycin) or genetic inhibition (BECLIN1 knockdown) of autophagy impairs apoptosis induced by Nutlin 3a.

Topics: Adenylate Kinase; Autophagy; Blotting, Western; Cell Line, Tumor; Enzyme Activation; Flow Cytometry; Humans; Imidazoles; Lentivirus; Leukemia; Microscopy, Confocal; Microscopy, Electron, Transmission; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

To elucidate new molecular mechanisms able to downregulate the mRNA levels of key oncogenes, such as B-Myb and E2F1, in a therapeutic perspective.. B-Myb and E2F1 mRNA levels were evaluated in primary B chronic lymphocytic leukemia (B-CLL, n = 10) and acute myeloid leukemia (AML, n = 5) patient cells, in a variety of p53(wild-type) and p53(mutated/deleted) leukemic cell lines, as well as in primary endothelial cells and fibroblasts. Knockdown experiments with siRNA for p53 and E2F1 and overexpression experiments with miR34a were conducted to elucidate the role of these pathways in promoting B-Myb downregulation.. In vitro exposure to Nutlin-3, a nongenotoxic activator of p53, variably downregulated the expression of B-Myb in primary leukemic cells and in p53(wild-type) myeloid (OCI, MOLM) and lymphoblastoid (SKW6.4, EHEB) but not in p53(mutated) (NB4, BJAB, MAVER) or p53(deleted) (HL-60) leukemic cell lines. The transcriptional repression of B-Myb was also observed in primary normal endothelial cells and fibroblasts. B-Myb downregulation played a critical role in the cell-cycle block in G(1) phase induced by Nutlin-3, as shown by transfection experiments with specific siRNA. Moreover, we have provided experimental evidence suggesting that miR-34a is a central mediator in the repression of B-Myb both directly and through E2F1.. Owing to the role of B-Myb and E2F1 transcription factors in controlling cell-cycle progression of leukemic cells, the downregulation of these oncogenes by miR-34a suggests the usefulness of therapeutic approaches aimed to modulate the levels of miR-34a.

Topics: Base Sequence; Cell Cycle Proteins; Cell Line, Tumor; Cells, Cultured; Down-Regulation; E2F1 Transcription Factor; Gene Expression Regulation, Leukemic; HCT116 Cells; HL-60 Cells; Humans; Imidazoles; Leukemia; MicroRNAs; Models, Biological; Oncogenes; Piperazines; Sequence Homology, Nucleic Acid; Trans-Activators; Transfection

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Death; Drug Screening Assays, Antitumor; Humans; Imidazoles; Leukemia; Phosphorylcholine; Piperazines; Tumor Cells, Cultured