PPAR-γ Agonists Induce Growth Arrest of Kit/AML1-ETO Leukemia Cells Acting on Diverse Pathways

Translocation 8;21, t(8;21), with an incidence of up to 12% is among the most common genetic aberrations found in Acute Myeloid leukemias (AMLs). t(8;21) rearranges the AML1 gene on chromosome 21 and the ETO gene on chromosome 8, generating an AML1-ETO fusion protein. It acts as dominant-negative repressor of genes involved in cell cycle control and myeloid differentiation. Despite its critical role in tumor initiation, AML1-ETO is not sufficient, per se, to cause an overt leukemia. It has to cooperate with other genetic alterations, such as those involving receptor tyrosine kinases. KIT tyrosine kinase gain-of-function mutations have been detected in about 50% of t(8;21) AMLs.

Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) agonists are known to inhibit proliferation and induce apoptosis in cancer cells, including those from hematologic malignancies. Despite several promising pre-clinical studies, results of clinical trials are not conclusive so far.

We investigated the effects and mechanisms of action of some PPAR-γ agonists in an AML1-ETO/Kit activated leukemia cell line: Kasumi-1. Troglitazone (TGZ) and rosiglitazone (RGZ) reduced proliferation rate and clonogenic potential of Kasumi-1, inducing G1 arrest and apoptosis in a dose and time dependent manner. Activated Kit induces proliferation mostly through the Src kinases/Ras/MAPK pathway. Erk1/2 is constitutively phosphorylated in Kasumi-1 cells and exposure to troglitazone (50μM) significantly reduced its phosphorylated state even if not as much as imatinib. The Kit signaling cascades include activation of PI3-kinase/AKT pathway, but surprisingly TGZ treatment did not affect AKT phosphorylation.

It has been shown that PPAR agonists 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid (CDDO) and 15-Deoxy-Δ12,14-ProstaglandinJ2 (15d-PGJ2) are able to suppress TGFβ-induced phosphorylation of Akt^S473, leading an anti-fibrotic activity in the lung. CDDO and 15d-PGJ2 inhibition effect have been demonstrated direct, PPAR-γindependent and mostly related to electrophilic carbons present in their structures. In the same cellular context rosiglitazone was found relatively poorly effective. We compared, in Kasumi-1 cells, activity of 15d-PGJ2 and CAY10410, two PPAR agonists with similar structure but while 15d-PGJ2 has an electrophilic center CAY10410 does not. CAY10410 is a strong PPAR agonist but was unable to induce any reduction in Kasumi-1 cells proliferation rate or induce apoptosis whereas 15d-PGJ2 strongly inhibited Kasumi-1 growth and induced apoptosis even more efficiently than troglitazone. Molecular characterization of such functional effect is of extreme interest and is ongoing.

TGZ and RGZ induced up-regulation of p27^Kip1 and p21^cip1. We found that it occurs by reduced proteosomal degradation and accumulation of p27^kip1 due to up-regulation of Connexin 43 (Cx43) and consequent downregulation of Skp2. AML1-ETO has been reported as Cx43 transcriptional inducer. Upregulation of p21^Waf1 seems to be, in preliminary experiments, mostly transcriptional and AML1-ETO direct induction of p21^Waf1has been described as well. Interestingly TGZ seems to restore AML1-ETO functions counteracted upon Kit constitutive activation.

Some, if not all, the effects might be receptor independent since they were just minimally lessened by a pre-treatment with high dose of GW9662, an irreversible PPAR-γ antagonist that covalently binds to the active site of PPAR-γ.

The overall prognosis of AML patients remains severe and AML incidence is expected to increase with the aging population, forcing the need for less toxic regimens in patients with co-morbid conditions precluding intensive chemotherapy. PPAR agonists are promising candidates as specific anticancer drugs within low toxicity multidrug regimens. However, the effectiveness of PPAR ligands seems to vary as various compounds, targeting distinct leukemic cells, behavior differently, because different are the leading molecular mechanism evoked. Their precise clarification will define the real extent of PPAR agonists’ therapeutic potential.