8-CPT-cAMP/all-trans retinoic acid targets t(11;17) acute promyelocytic leukemia through enhanced cell differentiation and PLZF/RARα degradation

Abstract

The refractoriness of acute promyelocytic leukemia (APL) with t(11;17)(q23;q21) to all-trans retinoic acid (ATRA)-based therapy concerns clinicians and intrigues basic researchers. By using a murine leukemic model carrying both promyelocytic leukemia zinc finger/retinoic acid receptor-α (PLZF/RARα) and RARα/PLZF fusion genes, we discovered that 8-chlorophenylthio adenosine-3', 5'-cyclic monophosphate (8-CPT-cAMP) enhances cellular differentiation and improves gene trans-activation by ATRA in leukemic blasts. Mechanistically, in combination with ATRA, 8-CPT-cAMP activates PKA, causing phosphorylation of PLZF/RARα at Ser765 and resulting in increased dissociation of the silencing mediator for retinoic acid and thyroid hormone receptors/nuclear receptor corepressor from PLZF/RARα. This process results in changes of local chromatin and transcriptional reactivation of the retinoic acid pathway in leukemic cells. Meanwhile, 8-CPT-cAMP also potentiated ATRA-induced degradation of PLZF/RARα through its Ser765 phosphorylation. In vivo treatment of the t(11;17) APL mouse model demonstrated that 8-CPT-cAMP could significantly improve the therapeutic effect of ATRA by targeting a leukemia-initiating cell activity. This combined therapy, which induces enhanced differentiation and oncoprotein degradation, may benefit t(11;17) APL patients.

Fig. 1.

The 8-CPT-cAMP/ATRA combined treatment induces APL cells differentiation. (A) NBT reduction assays of three U937 cell strains posttreatment of 8-CPT-cAMP (100 μM) and/or ATRA (1 μM) for 3, 5, and 7 d, respectively. (B) Morphological analysis of U937-mt-PLZF/RARα cells after 8-CPT-cAMP (100 μM) and/or ATRA (1 μM) treatment for 7 d (magnification: 1,000×). (C) Activities of pRARE-tk-Luc reporter transfected in both U937-mt-P/R9 and U937-mt-PLZF/RARα cells were detected after 8-CPT-cAMP (100 μM) and/or ATRA (1 μM) treatment for 24 h. (D) Real-time RT-PCR quantified the mRNA expression of RARα2, RARβ2, CEBPɛ, and TGM2 in U937-mt-PLZF/RARα cells after 8-CPT-cAMP (100 μM) and/or ATRA (1 μM) treatment for 24 and 48 h, respectively. Results were presented as mean ± SD of three independent experiments. Briefly, *P < 0.05, **P < 0.01, and ***P < 0.001 in comparison.

Fig. 2.

8-CPT-cAMP facilitates SMRT disassociation from PLZF/RARα through its Ser765 phosphorylation by activating the PKA pathway. (A) Schematic illustration of PLZF/RARα protein structure. Several potential sites were denoted in the corresponding domains of the fusion protein, and possible upstream regulators were also annotated aside. (B) U937-mt-PLZF/RARα cells were exposed to 100 μM ZnSO₄ for 12 h before treatment with 8-CPT-cAMP (100 μM) and/or ATRA (1 μM) for additional 24 h. Serine/threonine-specific phosphorylation level of PKA downstream substrates was assessed by Western blot (Upper). Arrows indicate the bands specific to the antibodies. Equal loading of protein was assessed with β-actin (Lower). (C) Mammalian two-hybrid analysis of PLZF/RARα-WT (Upper) or S765A mutant (Lower) with SMRT was done when exposed to different drug treatments. COS-7 cells were transiently transfected with plasmids, as indicated in SI Materials and Methods for 18 hours, and then 8-CPT-cAMP and/or ATRA were added for additional 6 hours. (D) ChIP assays in U937-mt-PLZF/RARα cells treated with indicated agents for 24 h. Briefly, *P < 0.05; N.S. refers to “not significant” in comparison by Student t test.

Fig. 3.

8-CPT-cAMP enhances ATRA-induced degradation of PLZF/RARα proteins through its Ser765 phosphorylation by PKA pathway activation. (A) A time course of PLZF/RARα expression in U937-mt-PLZF/RARα cells was determined at both mRNA and protein level after 8-CPT-cAMP and/or ATRA treatment. The cells were initially induced by 100 μM ZnSO₄ before treatment with 8-CPT-cAMP (100 μM) and/or ATRA (1 μM) for 12, 24, and 48 h, respectively. Nuclear proteins were extracted and detected by anti-RARα antibody. (B) Zn²⁺ induced-U937-mt-PLZF/RARα cells were preincubated with pan-caspases inhibitor z-VAD-fmk (40 μM), PKA pathway inhibitor H89 (10 μM), lysosome inhibitor chloroquine (100 μM), proteasome inhibitor MG132 (1 μM) for 2 h, and then treated with 8-CPT-cAMP (100 μM) and/or ATRA (1 μM) for an additional 24 h. PLZF/RARα and Lamin B proteins were examined by Western blot. (C) 293T cells transiently expressing pVP16-PLZF/RARα-WT and S765A proteins were treated by 8-CPT-cAMP (100 μM) and/or ATRA (1 μM) for 24 h. Both wild-type and mutant PLZF/RARα were immunoblotted by anti-RARα antibody. Equal loading was assessed by Lamin B or β-actin expression. Abbreviations: +/− Zn, with or without ZnSO₄; Un, untreated; C, 8-CPT-cAMP; A, ATRA; zVAD, z-VAD-fmk; Chl, chloroquine; Veh, vehicle.

Fig. 4.

In vivo 8-CPT-cAMP/ATRA combined treatment improves the survival of t(11;17) APL mice by targeting of LICs. (A) Scheme of the long-term drug treatment in vivo and secondary BM transplantations (BMT). (B) Kaplan–Meier survival curve of primary mice under different drug treatments. (C) Spleen index of primary mice on day 27 (i) and the survival curve of their secondary recipients inoculated with 1,000 LICs (Mac-1⁺/Gr-1⁺/c-Kit⁺) each (ii). (D) Spleen index of primary mice on day 41 (i) and the survival curve of their secondary recipients inoculated with 1,000 LICs (Mac-1⁺/Gr-1⁺/c-Kit⁺) each (ii).