Retinoic acid responsiveness and resistance in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by reciprocal chromosomal translocations invariably involving the retinoic acid receptor alpha (RARalpha) gene on chromosome 17. In the vast majority of APL cases, the RARalpha gene is fused to the PML gene on chromosome 15, generating the fusion PML-RARalpha. The chimeric PML-RARalpha protein plays a critical role in APL leukemogenesis. Retinoic acid (RA) can overcome the differentiation block conferred by PML-RARalpha and induce APL cells to terminally maturate into granulocytes. However, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones derived from the human APL cell line, NB4. These resistant subclones display altered RA-binding and reduced transactivation of retinoid response elements (RAREs) upon RA treatment. In the subclone R4, we identified a point mutation changing a leucine to praline in the ligand binding domain of the fusion PML-RARalpha protein that causes a loss of ligand binding. The mutant PML-RARalpha protein retains its ability to heterodimerize with RXRalpha and bind to RAREs. Thus, it functions as a dominant negative transcriptional inhibitor of the coexpressed wild-type RARalpha. The mutant PML-RARalpha has impaired RA-dependent interaction with receptor coregulatory factors, SMRT and ACTR. Histone deacetylase inhibitor, Trichostatin A (TSA), in combination with RA can overcome the dominant negative activity of the mutant PML-RARalpha in R4 and relieve transcriptional inhibition on RAREs. Further, TSA potentiates and partially restores RA-induced differentiation of NB4 and R4 cells respectively. When we examined the interaction of specific cofactors with retinoid receptors in NB4 cells compared to that in RA-resistant cell lines, we isolated the DRIP/TRAP protein complex. The ligand-inducible interaction between the DRIP/TRAP complex and retinoid receptors is independent of RA sensitivity and the expression of PML-RARalpha, supporting the global role of DRIP/TRAP in transcriptional regulation. Due to the frequent occurrence of RA-resistance in APL patients, we investigated the effects of arsenic trioxide (As2O3) in APL cells. As2O3 induces apoptosis and loss of the PML-RARalpha fusion protein in RA-sensitive as well as RA-resistant APL cells. As2O3 and RA induce different patterns of gene regulation, and they inhibit the phenotype induced by each other. In summary, we demonstrated that mutation in the ligand binding domain of the PML-RARalpha fusion protein accounts for an important mechanism underlying RA resistance. We also showed that inhibitors of histone deacetylase and As2O3 have potential clinical applications in treating relapsed APL patients and other human leukemias.