In vivo activation of cAMP signaling induces growth arrest and differentiation in acute promyelocytic leukemia

Abstract

Differentiation therapy for acute myeloid leukemia uses transcriptional modulators to reprogram cancer cells. The most relevant clinical example is acute promyelocytic leukemia (APL), which responds dramatically to either retinoic acid (RA) or arsenic trioxide (As(2)O(3)). In many myeloid leukemia cell lines, cyclic adenosine monophosphate (cAMP) triggers growth arrest, cell death, or differentiation, often in synergy with RA. Nevertheless, the toxicity of cAMP derivatives and lack of suitable models has hampered trials designed to assess the in vivo relevance of theses observations. We show that, in an APL cell line, cAMP analogs blocked cell growth and unraveled As(2)O(3)-triggered differentiation. Similarly, in RA-sensitive or RA-resistant mouse models of APL, continuous infusions of 8-chloro-cyclic adenosine monophosphate (8-Cl-cAMP) triggered major growth arrest, greatly enhanced both spontaneous and RA- or As(2)O(3)-induced differentiation and accelerated the restoration of normal hematopoiesis. Theophylline, a well-tolerated phosphodiesterase inhibitor which stabilizes endogenous cAMP, also impaired APL growth and enhanced spontaneous or As(2)O(3)-triggered cell differentiation in vivo. Accordingly, in an APL patient resistant to combined RA-As(2)O(3) therapy, theophylline induced blast clearance and restored normal hematopoiesis. Taken together, these results demonstrate that in vivo activation of cAMP signaling contributes to APL clearance, independently of its RA-sensitivity, thus raising hopes that other myeloid leukemias may benefit from this therapeutic approach.

Figure 1.

8-Cl-cAMP induces growth arrest in RA-sensitive (RA^S, a) or RA-resistant (RA^R, b) APL in mice. (Top) Spleen weight after 7 d of 8-Cl-cAMP treatment or no therapy (Φ). (Middle) Bone marrow sample after May-Grünwald Giemsa staining. Note an apoptotic cell (arrow) in the treated sample. (Bottom) Liver from the same mice.

Figure 2.

Arsenic and cAMP synergize to induce tumor regression and differentiation in RA-sensitive APL. (a) 8-CPT-cAMP potentiates As₂O₃-induced NBT reduction at 4 d in the APL cell line NB4. (b) In a mouse model of APL, continuous infusion of 8-Cl-cAMP for 3 d greatly reduces spleen weight and synergizes with As₂O₃ (As) to induce tumor reduction (one representative experiment). (c) CD11b expression on bone marrow cells after 24 h of in vivo treatments, as indicated. (d) 8-Cl-cAMP induces p21 expression in leukemic bone marrow after 24 h of in vivo treatment. *Cross-reactive protein. (e and f) 8-Cl-cAMP synergizes with As₂O₃ to eradicate leukemia. 3 d of combined treatment restores normal hematopoiesis in the bone marrow (e) and induces tumor clearance from the liver (f). (g) Theophylline (T) therapy (3 d) induces blast differentiation. Note the synergistic action of As.

Figure 3.

RA and cAMP synergize to induce tumor regression and differentiation. (a) Spleen weight after 7 d of treatment or untreated (Φ). (b) Corresponding bone marrow morphology.

Figure 4.

cAMP synergizes with RA in RA-resistant APL. (a) Spleen weight after 7 d of treatment. (b) Corresponding bone marrow morphology. Two examples of marrows from mice exposed to RA and cAMP are shown.

Figure 5.

Theophylline synergizes with RA/As₂O₃ therapy in a multirelapsed patient with RA/As₂O₃-resistant APL. (a) Schematic representation of clinical events. (b and c) Representative images of bone marrow samples taken at days 0, 14, and 28 of the initial RA/As₂O₃ course (b) or the following RA/As₂O₃/theophylline course (c). Note the rapid differentiation in (c) with the reappearance of normal erythroblasts at day 14, as well as the maintenance of normal hematopoiesis despite leukemia relapse. Hb, hemoglobin; Plt, platelet; WBC, white blood cell.