IntroductionRetinoic acid receptors (RARs) are ligand-dependent transcription factors that function in heterodimeric states involving retinoid X receptors (RXRs). Both RARs and RXRs exist in 3 isoforms, ␣,  and ␥, and they are the master regulators of a plethora of physiological processes, such as embryo development, organ homeostasis, and cellular events such as proliferation, differentiation, and death. 1,2 Almost all cases of acute promyelocytic leukemia (APL), a unique subtype of acute myeloid leukemia (AML), express oncogenic fusion protein involving the RAR-␣ gene due to chromosome translocations. 3 A typical example is the promyelocytic leukemia (PML)-RAR-␣ fusion protein that is formed as a result of the t(15;17) translocation found in more than 98% of APL patients. 4,5 All-trans retinoic acid (ATRA), a natural ligand for the RARs, induces complete remission in almost all PML-RAR-␣-positive APL patients through the induction of terminal differentiation and/or eradication of leukemia-initiating cells (LICs) by PML-RAR-␣ destruction. [6][7][8] However, the therapeutic use of ATRA is not free of concerns. Notably, the remission in patients treated with ATRA alone is of short duration because of the rapid development of retinoid resistance. 9 Moreover, the clinical effectiveness of ATRA is limited to t(15;17)-positive APL patients. It is believed that one possible means to overcome these problems might be the combination of ATRA with other agents, such as arsenic trioxide (As 2 O 3 ). 7,10,11 On the other hand, substantial investigations suggest that RARs and RXRs are also useful drug targets for the treatment of cancer and metabolic diseases, such as diabetes and obesity. 12 The success of RAR modulation in the treatment of APL has generated considerable interests in the development of RAR/RXR modulators for these diseases, as reviewed by Altucci et al. 13 By screening a series of natural compounds, here we show that pharicin B, a novel natural ent-kaurene diterpenoid from Isodon pharicus leaves, can stabilize RAR-␣ protein in the presence and absence of ATRA and enhance the differentiation-inducing effect of ATRA in several AML cell lines and some primary leukemic cells, especially those from APL patients. Moreover, pharicin B can overcome retinoid resistance in some NB4-derived, ATRA-resistant subclones. However, pharicin B can also stabilize PML-RAR-␣ protein in APL cells, the catabolism of which is regarded as crucial for LIC eradication and long-term remission of mouse APL by ATRA and/or As 2 O 3 . 7,14 Collectively, these findings suggest that pharicin B is an extraordinary tool to probe potential pathophysiological and therapeutic roles of RAR-␣ and PML-RAR-␣. In addition, an approach combining ATRA with pharicin B warrants further investigation as a therapeutic strategy for myeloid leukemia.
Methods
Patients and cell linesBone marrow samples were collected from 12 cases of newly diagnosed AML patients at Rui-Jin and Ren-Ji Hospitals affiliated to Shanghai Jiao were cultured in RPMI 1640 medium (Si...