The sorafenib plus nutlin-3 combination promotes synergistic cytotoxicity in acute myeloid leukemic cells irrespectively of FLT3 and p53 status.
BACKGROUND
Both the multi-kinase inhibitor sorafenib and the small molecule inhibitor of the MDM2/p53 interaction, nutlin-3, used alone, have shown promising anti-leukemic activity in acute myeloid leukemia cells. Thus, in this study we investigated the effect of the combination of sorafenib plus nutlin-3 in acute myeloid leukemia.


METHODS
Primary acute myeloid leukemia blasts (n=13) and FLT3(wild-type)/p53(wild-type) (OCI-AML3), FLT3(mutated)/p53(wild-type) (MOLM), FLT3(mutated)/p53(mutated) (MV4-11), FLT3(wild-type)/p53(deleted) (HL60) or FLT3(wild-type)/p53(mutated) (NB4) acute myeloid cell lines were exposed to sorafenib, used alone or in association with nutlin-3 at a 1:1 ratio, in a range of clinically achievable concentrations (1-10 μM). Induction of apoptosis and autophagy was evaluated by transmission electron microscopy and by specific flow cytometry analyses. The levels of Mcl-1, p53 and Bak proteins were analyzed by western blotting. Knock-down of Bax and Bak gene expression was performed in transfection experiments with specific short interfering RNA.


RESULTS
The sorafenib+nutlin-3 drug combination exhibits synergistic cytotoxicity in primary acute myeloid leukemia blasts and in acute myeloid leukemia cell lines with maximal cytotoxicity in FLT3(mutated) MV4-11 and MOLM, followed by the FLT3(wild-type) OCI-AML3, HL60 and NB4 cell lines. The cytotoxic activity of sorafenib+nutlin-3 was characterized by an increase of both apoptosis and autophagy. Moreover, Bax and Bak showed prominent roles in mediating the decrease of cell viability in response to the drug combination in p53(wild-type) OCI-AML3 and p53(deleted) HL-60 cells, respectively, as demonstrated in transfection experiments performed with specific short interfering RNA.


CONCLUSIONS
Our data demonstrate that acute myeloid leukemia cells show a variable but overall good susceptibility to the innovative therapeutic combination of sorafenib+nutlin-3, which differentially involves the pro-apoptotic Bcl-2 family members Bax and Bak in p53(wild-type) and p53(deleted) cells.
Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy. giorgio.zauli@unife.it
Giorgio
G
Zauli
Claudio
C
Celeghini
Elisabetta
E
Melloni
Rebecca
R
Voltan
Manuele
M
Ongari
Mario
M
Tiribelli
Maria Grazia
MG
di Iasio
Francesco
F
Lanza
Paola
P
Secchiero
D000970:Antineoplastic Agents / Q000819:agonists / Q000494:pharmacology*; D004357:Drug Synergism; D005260:Female; D018922:HL-60 Cells; D006801:Humans; D007093:Imidazoles / Q000819:agonists / Q000494:pharmacology*; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D009536:Niacinamide / Q000819:agonists / Q000031:analogs & derivatives* / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000819:agonists / Q000494:pharmacology*; D010879:Piperazines / Q000819:agonists / Q000494:pharmacology*; D000077157:Sorafenib; D016159:Tumor Suppressor Protein p53; D051941:fms-Like Tyrosine Kinase 3
D000970:Antineoplastic Agents; D007093:Imidazoles; D010671:Phenylurea Compounds; D010879:Piperazines; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D009536:Niacinamide; C482205:nutlin 3; D000077157:Sorafenib; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3