Acute promyelocytic leukemia (APL) is a myeloid leukemia containing the t(15;17) rearrangement that expresses the oncogenic PML/RARα fusion protein. Expression of PML/RARα is also required for clinical remissions of APL cases to all-trans-retinoic acid (RA). RA response in APL is linked to the proteasomal degradation of PML/RARα. Yet, the exact mechanisms responsible are not well understood. The present study comprehensively explored RA effects on PML/RARα degradation as a strategy to understand precisely therapeutic effects of RA in APL. Constructs containing different PML/RARα domains were transiently transfected into BEAS-2B human bronchial epithelial cells that do not express PML/RARα. RA effects on expression of PML/RARα domains were analyzed by immunoblotting. Results demonstrate that RA confers a dose and time-dependent decrease in expression of PML/RARα and the RARα domain of this fusion protein. In contrast, no effect was observed on the PML domain. Thus, RA selectively targets RARα to degrade PML/RARα protein. We previously reported that RA treatment also induces UBE1L, an E1-like ubiquitin-activating enzyme. This was found as directly responsible for PML/RARα degradation. Co-transfection of UBE1L with different PML/RARα domains resulted in degradation of PML/RARα and the PML (but not RARα) domain of this fusion protein. Thus, RA-activation of UBE1L targets PML/RARα for degradation, but at a site different from that affected by RA. Notably, UBP43, the ISG15 specific deconjugase that opposes UBE1L effects, reverses UBE1L-dependent, but not RA-dependent degradation of PML/RARα. The reversible proteasomal inhibitor, ALLN, inhibited UBE1L and RA effects. These data reveal that retinoid-mediated PML/RARα degradation is proteasome-dependent, but that RA engages a biochemically distinct pathway that also leads to PML/RARα degradation in APL. Studies are underway to uncover PML/RARα residues required for this degradation. Taken together, these findings establish that RA degrades different domains of the oncogenic PML/RARα product through distinct mechanisms. UBE1L is also able to engage the repression of other oncogenic proteins. Since UBE1L expression is often deregulated in specific epithelial cancers, this novel degradation program has biological relevance in targeting cancers beyond APL.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]