In vitro characterization of the acute promyelocytic leukemia variant fusion oncoprotein NuMA-RARalpha

Acute promyelocytic leukemia (APL) is characterized by the presence of gene rearrangements involving the retinoic acid receptor a (RARalpha) gene locus on chromosome 17. These rearrangements result in the formation of X-RARalpha fusion oncoproteins which act as aberrant transcriptional repressors, thus making APL a model disease in which to study the role of aberrant transcriptional repression in tumongenesis. Our group identified two variant X-RARalpha---NPM-RARalpha and NuMA-RARalpha---during the last decade. Here, we characterize these variant fusion proteins, in comparison with PML-RARa and PLZF-RARalpha, in terms of their DNA binding properties and ability to repress transcription from the retinoic acid and peroxisome proliferator response elements. Our data suggest a role for X-RARalpha in inhibiting signaling through the PPARgamma/PPRE pathway, consistent with recent evidence for the deregulation of pathways external to retinoid signaling during APL pathogenesis. Our data also indicate that X-RARalpha fusions may fall into two distinct classes in their ability to bind to the RARE and PPRE and to activate ligand-responsive genes. Furthermore, we show that NuMA-RARalpha is capable of causing ligand-dependent transcriptional super-activation at both RAREs and PPREs, exhibiting a property that is unique among the X-RARalpha fusion proteins. We went on to develop the U937/NuMA-RARalpha stable cell line, the first cell culture system expressing this fusion. We report that NuMA-RARalpha was stably expressed at the protein level, and found in both the cytoplasm and nucleus. Furthermore, NuMA-RARalpha did not appear to interfere with localization of NuMA to the mitotic spindle. We found that the PPARgamma ligand troglitazone (TZ) can inhibit the cellular proliferation of cells expressing NuMA-RARa by induction of apoptosis. We validated these findings in an ex vivo system, by demonstrating induction of apoptosis in hCG-NuMA-RARa transgenic mouse LTMCs in response to TZ. Apoptosis in these cells is due to activation of both receptor-mediated and mitochondrial pathways, operating through caspases-8 and -9. This effect of troglitazone was not limited to NuMA-RARalpha, and was not species or cell type specific, thus implicating it as a possible alternative therapy in APL.

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