Although TP53 mutations are rare in acute myeloid leukemia (AML), inactivation of wild-type p53 protein frequently occurs through overexpression of its negative regulator MDM2 (murine double minute 2). We investigated the effects of MI-63, a small molecule that activates p53 by inhibition of MDM2-p53 interaction [ Ki value of 3 nM (

J Med Chem.
2006
;
49
(12):
3432
–5
)] in AML cell lines. Treatment with MI-63 triggered apoptosis (evidenced by loss of membrane potential and externalization of phosphatidylserine) in AML cell lines with wild-type p53 (OCI-AML-3 and MOLM13) in a time and concentration-dependent manner (IC50 at 72 hrs.= 2.5 μM for OCI-AML-3 and 1 μM for MOLM-13), while a p53-null AML cell line (HL-60) was resistant (IC50 not reached at 10 μM). Moreover, knockdown of p53 in OCI-AML3 cells rendered this cell line resistant to MI-63 induced apoptosis while control vector infected OCI-AML-3 cells remained as sensitive to MI-63 similar to the parental cells.

Mechanistic studies showed that MI-63 blocks G1/S phase transition in AML cells with wild-type p53 resulting in accumulation of cells in G1 phase (percentage cells inG1 phase at 24 hrs. = 88.66% vs 43.49% in cultures with DMSO control) while MI-61, a skeletally related but inactive control compound failed to do so (41.63%). Treatment with MI-63 increased cellular levels of p53 and p53 dependent proteins in OCI-AML-3 cells that include p21 and BH3-only pro-apoptotic protein Puma and pro-apoptotic multi-domain Bcl-2 family member Bax. Additionally, MI-63 induced a profound decrease in the levels of MDM4, an MDM2 homolog that has been reported to mediate resistance to the effects of nutlin-3a, suggesting that MI-63 may offer a therapeutic advantage in cells expressing high levels of MDM4.

Finally, supporting the concept that increased levels of p53 modulate the apoptotic rheostat both directly, by behaving as a BH3-only protein, and indirectly by increasing the levels of sensitizer BH3-only proteins, MI-63 potently synergized with AT-101, an orally available pan inhibitor of Bcl-2, Bcl-xL and Mcl-1 (currently being evaluated as an antitumor agent in Phase I/II trials by Ascenta Therapeutics), to induce mitochondrial dysfunction and apoptosis in OCI-AML-3 cells (average combination index = 0.055±0.019).

Taken together our results support preclinical evaluation of novel small molecule MI-63 alone and in combination with Bcl-2 inhibitors for the therapy of AML. The studies in primary AML samples are ongoing.

Fig.1:
Fig.1:. MI-63 Induced Apoptosis Requires Intact p53
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MI-63 Induced Apoptosis Requires Intact p53

Fig.1:
Fig.1:. MI-63 Induced Apoptosis Requires Intact p53
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MI-63 Induced Apoptosis Requires Intact p53

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Fig.2:
Fig.2:. Efect of MI-63 on p53 and Related Proteins (comparison with N3a, a known MDM2 inhibitor included)
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Efect of MI-63 on p53 and Related Proteins (comparison with N3a, a known MDM2 inhibitor included)

Fig.2:
Fig.2:. Efect of MI-63 on p53 and Related Proteins (comparison with N3a, a known MDM2 inhibitor included)
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Efect of MI-63 on p53 and Related Proteins (comparison with N3a, a known MDM2 inhibitor included)

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Topics:
antineoplastic agents, apoptosis, bcl-xl protein, cell lines, dimethyl sulfoxide, leukemia, myelocytic, acute, mechlorethamine, phosphatidylserines, protein p53, small molecule

Disclosures: Dajun Yang is an employee of Ascenta Therapeutics, Inc.; Dajun Yang has ownership interest in Ascenta Therapeutics, Inc.

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Corresponding author

2006, The American Society of Hematology
2006
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