12-O-tetradecanoylphorbol-13-acetate (TPA) is a phorbol ester with the capacity to induce differentiation and/or apoptosis in multiple cell lines and primary cells [1–8]. Phorbol esters activate protein kinase C (PKC) and modulate the activity of multiple downstream cell signaling pathways, including the mitogen-activated protein kinase (MAPK) pathways [9–12].
The MAPKs are proline directed ser/thr kinases categorized into families based upon sequence homologies [13–21]. Each MAPK phosphorylates downstream substrates, including kinases and transcription factors. Histones, chromatin components, and other transcriptional modulators are phosphorylated secondarily. The MAPK extracellular signal-regulated kinase (ERK) is often associated with cellular proliferation; ERK activation being central to signaling from receptor tyrosine kinases, G-protein coupled receptors, selected integrins, and activated ras and raf. In contrast, activation of c-Jun N terminal-kinases (JNK) and p38 kinases has been associated with stress responses and induction of apoptosis [13–21]. However, there are complex interactions among MAPKs and many factors, including the kinetics of specific MAPK activation and inactivation, can determine phenotypic effects [22–24].
Regulation of MAPK cascades is complex. The subcellular localization of individual kinases, the nature of associated ‘‘scaffolding‘‘/interacting proteins, the presence of single and dual specificity MAPK phosphatases which target specific MAPK isoforms, and the duration of kinase activation all modulate MAPK signaling [9–11, 22–26]. Hence, activation of specific MAPKs represents a balance of phosphorylation and dephosphorylation mediated at various subcellular locations in association with various interacting proteins.
The effects of TPA on MAPK pathways may be particularly relevant to the differentiating and proapoptotic effects of TPA in certain cells. For example, molecular analysis of AML indicates at least two overlapping classes of interacting genetic alterations. Signaling pathway alterations facilitate proliferation and survival, whereas transcription factor mutations/fusions often alter chromatin remodeling and inhibit differentiation (reviewed in reference ). Approximately 30% of AML samples contains a Flt 3 receptor that is constitutively active as a consequence of an internal tandem duplication (ITD) or kinase domain mutation and additional AML samples have mutations in other receptor protein tyrosine kinase genes (e.g., c-kit) or ras [27–29]. Furthermore, other related myeloid malignancies have translocations (e.g., BCR-ABL, ABL-TEL, TEL-PDGFR, TEL-JAK2) which result in fusion kinases that may activate ras and other signaling pathway components . The downstream effects of these alterations often include constitutive ERK activation, present in 51–70% of primary AML, including all samples containing the Flt 3 receptor ITD [30–32]. In addition, pharmacologic inhibition of ERK pathway activation results in growth inhibition of myeloid leukemia cell lines . Recent studies have identified TPA as an agent that modulates ERK activation in primary AML cells. In a subset of primary AML cells studied ex vivo, prolonged exposure to TPA reduced ERK activation at 24 h and induced apoptosis .
The capacity of TPA to induce phenotypic changes, characteristic of differentiation and/or apoptosis in hematopoietic cell lines, led investigators in China to undertake a pilot study of TPA in patients with myeloid malignancies . A variety of doses and schedules, in conjunction with cytarabine or vitamin D3, were used. Clinical efficacy, as indicated by reduction in bone marrow myeloblasts and/or improvement of blood counts, was apparent in several treated patients, and the most prominent adverse effects included fevers, chills, dyspnea, hematuria, and phlebitis. These adverse effects were transient and repeated cycles of treatment were administered to most patients. Although the adverse effects were not ascribed to individual components of each patient’s therapy, they were not characteristic of cytarabine or vitamin D3, and were felt to be most likely related to TPA.
A subsequent formal phase I study of TPA was initiated in the United States. TPA-induced alterations in
AML cell gene expression and immunophenotype in selected patient samples were reported after treatment of the first 14 patients . In addition, a biologic assay for TPA-like differentiating activity in the blood of treated patients was developed and used to determine the kinetics of TPA-like activity in the blood of five treated patients . A biologic assay was required because a physical assay of appropriate sensitivity could not be developed. This report describes the maximally tolerated dose and dose- limiting toxicity profile of TPA after the treatment in 35 patients and completion of the phase I trial.