nutlin-3a and Blast-Crisis

The Bcr-Abl tyrosine kinase regulates several Bcl-2 family proteins that confer resistance to apoptosis in chronic myeloid leukemia (CML) cells. Given p53's ability to modulate the expression and activity of Bcl-2 family members, we hypothesized that targeting Bcr-Abl, Bcl-2, and p53 concomitantly could have therapeutic benefits in blast crisis (BC) CML and in quiescent CML CD34+ cells that are insensitive to tyrosine kinase inhibitors (TKI). We examined the effects of the MDM2 inhibitor nutlin3a and its combination with the dual Bcl-2 and Bcl-xL inhibitor ABT-737, and the Bcr-Abl inhibitor nilotinib on BC CML patient samples. We found that in quiescent CD34+ progenitors, p53 expression is significantly lower, and MDM2 is higher, compared to their proliferating counterparts. Treatment with nutlin3a induced apoptosis in bulk and CD34+CD38- cells, and in both proliferating and quiescent CD34+ progenitor CML cells. Nutlin3a synergized with ABT-737 and nilotinib, in part by inducing pro-apoptotic, and suppressing anti-apoptotic, Bcl-2 proteins. Nilotinib inhibited the expression of Bcl-xL and Mcl-1 in BC CML cells. These results demonstrate that p53 activation by MDM2 blockade can sensitize BC CML cells, including quiescent CD34+ cells, to Bcl-2 inhibitor- and TKI-induced apoptosis. This novel strategy could be useful in the therapy of BC CML.

Topics: ADP-ribosyl Cyclase 1; Antigens, CD34; Antineoplastic Agents; Apoptosis; bcl-X Protein; Biphenyl Compounds; Blast Crisis; Cell Proliferation; Enzyme Activation; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Humans; Imatinib Mesylate; Imidazoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Glycoproteins; Nitrophenols; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Pyrimidines; Sulfonamides; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Activation of p53 by murine double minute (MDM2) antagonist nutlin-3a or inhibition of X-linked inhibitor of apoptosis (XIAP) induces apoptosis in acute myeloid leukemia (AML) cells. We demonstrate that concomitant inhibition of MDM2 by nutlin-3a and of XIAP by small molecule antagonists synergistically induced apoptosis in p53 wild-type OCI-AML3 and Molm13 cells. Knockdown of p53 by shRNA blunted the synergy, and down-regulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin-3a-induced apoptosis, suggesting that the synergy was mediated by p53 activation and XIAP inhibition. This is supported by data showing that inhibition of both MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Importantly, p53 activation and XIAP inhibition enhanced apoptosis in blasts from patients with primary AML, even when the cells were protected by stromal cells. Mechanistic studies demonstrated that XIAP inhibition potentiates p53-induced apoptosis by decreasing p53-induced p21 and that p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of second mitochondria-derived activator of caspases (SMAC) and by inducing the expression of caspase-6. Because both XIAP and p53 are presently being targeted in ongoing clinical trials in leukemia, the combination strategy holds promise for expedited translation into the clinic.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Blast Crisis; Carrier Proteins; Caspase 6; Cell Line, Tumor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Leukemic; Gene Knockdown Techniques; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; Mice; Mitochondrial Proteins; Piperazines; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Stromal Cells; Tumor Suppressor Protein p53; X-Linked Inhibitor of Apoptosis Protein

We have compared the cytotoxic/cytostatic responses of the SKW6.4 lymphoblastoid B-cells to the alkylating agent chlorambucil, the purine analog fludarabine, the non-genotoxic activator of the p53 pathway, Nutlin-3, used alone or in association with the death-inducing ligand recombinant TRAIL. Exposure to chlorambucil, fludarabine, and Nutlin-3 induced p53 accumulation and variably affected cell cycle progression in SKW6.4 lymphoblastoid cells. In particular, chlorambucil induced cell cycle accumulation at the G2/M checkpoint; Nutlin-3 induced early cell cycle arrest at the G1/S checkpoint, while fludarabine showed an intermediate behavior. On the other hand, recombinant TRAIL alone did not affect cell cycle progression but induced a rapid increase of apoptosis. Analysis of the gene expression profile of the p53-transcriptional targets showed distinct features between chlorambucil, Nutlin-3 and fludarabine, which likely account for their differential effect on cell cycle in SKW6.4 cells. In particular, chlorambucil upregulated the steady-state mRNA expression of SFN/14-3-3sigma, a gene involved in G2/M cell cycle arrest. Of note, all agonists upregulated TRAIL-R2 expression in SKW6.4 cells both at the mRNA and protein levels. Consistently, pretreatment with chlorambucil, fludarabine and Nutlin-3 enhanced SKW6.4 sensitivity to TRAIL-mediated apoptosis.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; B-Lymphocytes; Blast Crisis; Cell Cycle; Cell Line, Tumor; Chlorambucil; Humans; Imidazoles; Leukemia, Lymphocytic, Chronic, B-Cell; Piperazines; Proto-Oncogene Proteins c-mdm2; Receptors, TNF-Related Apoptosis-Inducing Ligand; Up-Regulation; Vidarabine