The EGFR-Inhibitor Erlotinib Induces Apoptosis in Neoplastic Cells of Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), an Effect Determined by the Expression Level of Nucleophosmin (NPM).

Abstract

Background: the epidermal-growth-factor-receptor (EGFR)-inhibitor erlotinib was rationally designed to antagonize the deregulated EGFR-activity in solid tumors. Abundant studies in these entities not only demonstrated clinical efficacy, but also a favorable toxicity profile. In particular the absence of hematopoietic toxicity prompted us to investigate the therapeutic potential of erlotinib in MDS and AML cells.Methods: We incubated ex vivo cells from patients with MDS (n=4, 2 lower risk and 2 higher risk) and AML (n=6, de novo: 3; post MDS: 3), as well as a broad spectrum of myeloid cell lines (P39, KG-1, HL-60, MV4-11, MOLM-13) with increasing dosages of erlotinib (1μM to 10μM). As controls (n=4) we used non-malignant CD34 + bone marrow cells. Before incubation, all ex vivo cells underwent CD34 + selection. Serial FACS-analyses of parameters determining apoptosis (DIOC/PI and AnnexinV/PI) were carried out over a maximum of 6 days.Results: We found that erlotinib was able to induce a considerable degree of apoptosis in MDS and AML cells. Although there was a high interindividual difference in sensitivity towards erlotinib, “responders” treated with 10μM erlotinib showed an increase of apoptotic cells between 20–30% after 72h, which reached a maximum of 60% on day 6. This apoptosis-inducing effect was achieved in a dose-dependent manner and not restricted to a specific entity. Noteworthy, erlotinib exhibited no toxicity towards non-neoplastic progenitor cells. Evaluating the molecular mechanisms determining sensitivity we showed that the apoptosis-inducing effect of erlotinib critically depended on the expression level of NPM. Thus erlotinib-resistant myeloid cell lines (i.e. P39) exhibited a higher epression of NPM than sensitive cell lines (i.e. KG-1). In addition, down-regulation of NPM by small-interfering RNA not only increased the apoptosis-inducing effect of erlotinib in sensitive cells, but moreover established sensitivity in otherwise erlotinib-resistant cells. Accordingly, siRNA-induced down-regulation of NPM in P39 cells elevated the percentage of apoptotic cells upon treatment with 10μM erlotinib by about 30% as compared to mock-transfected controls. Conclusion, we showed an off-target effect of erlotinib, as evidenced by its ability to induce apoptosis in EGFR-negative cells. Of particular interest is the observation that erlotinib induced apoptosis exclusively in neoplastic myeloid cells while sparing non-malignant progenitors. To the best of our knowledge, this is the first report providing evidence for the therapeutic potential of erlotinib in MDS and AML.