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Induction of AML Stem Cell-Specific Apoptosis Using a Proteasome Inhibitor/Anthracycline Combination Is Mediated by NFkappaB Inhibition and p53 Activation

Guzman, M.L.; Swiderski, C.F.; Rossi, R.M.; Grimes, B.A.; Howard, D.S.; Szilvassy, S.J.; Jordan, C.T.

Blood 100(11): Abstract No. 2161

2002


ISSN/ISBN: 0006-4971
Accession: 035124553

Studies have shown that human acute myelogenous leukemia (AML) originates from a rare population of leukemic stem cells (LSCs). LSCs are found in nearly all AML subtypes and are sufficient to initiate and maintain leukemic growth in both long-term cultures and NOD/SCID mice. LSCs share many characteristics with normal hematopoietic stem cells (HSC), including the ability to self-renew and a predominantly G0 cell cycle status. Since conventional chemotherapy drugs typically target actively cycling leukemic blast cells, the quiescent LSC population is likely to be drug resistant. Recent studies have described unique molecular differences between normal and leukemic stem cells. In particular, NFkappaB was found to be constitutively active in primitive AML cells, but inactive in normal HSCs. We thus hypothesized that the unique properties of leukemic cells could be exploited to induce apoptosis in the LSC population while sparing normal stem cells. To test this hypothesis, we utilized a novel strategy using treatment of primary AML cells with the proteasome inhibitor MG-132 and the anthracycline Idarubicin. Primary AML cells treated with 0.25 muM MG-132 in combination with 15ng/ml IDR showed a rapid decrease in cell viability (viable cell average=11% +/- 5% in 12h) and also a decrease of NF-kappaB binding activity. Importantly, induction of apoptosis was readily evident in the primitive LSC population, as well as in more mature AML cells. In contrast, normal primitive hematopoietic cells obtained from cord blood (CB) did not undergo apoptosis upon MG-132/IDR treatment (viable cell average= 73% +/- 9%). Further, AML cells treated with MG-132/IDR for as little as 12h demonstrated a dramatic reduction of engraftment in the NOD/SCID mouse xenotransplant model. In contrast, the engraftment ability of identically treated CB samples was not affected. To determine the role of NF-kappaB in AML cell survival, we performed gene expression analyses and molecular genetic studies using an adenovirus vector encoding a dominant negative allele of IkappaBalpha (Ad-dnIkappaBalpha), which is known to specifically inhibit NF-kappaB. High-density gene expression arrays of Ad-dnIkappaBalpha infected cells revealed the consistent down-regulation of NF-kappaB regulated genes. In contrast to the 89% cell death seen with MG-132/IDR 12h treatment, infection with Ad-dnIkappaBalpha resulted in only 50% cell death at 36h post-infection. This observation indicates that while NF-kappaB signaling clearly affects AML cell survival, it is not sufficient alone to mediate the rapid apoptotic response seen with MG-132/IDR. Similar results were obtained with samples from blast crisis CML. To characterize other critical components of apoptosis, we analyzed the activation of p53, as determined by RNase protection and immunoblot assays. The treatment with MG-132/IDR resulted in elevated p53 protein levels and increased transcription of p53 target genes, such as p21, GADD45 and bax. These data strongly suggest that use of the proteasome inhibitor MG-132 in combination with Idarubicin can successfully target the primitive AML compartment, which is generally resistant to typical chemotherapeutic treatments. Equally important, this drug combination does not appear to harm the normal hematopoietic compartments and could therefore provide new treatment options for AML.

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