Abstract

Background: The immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), consisting of an anti-CD33 antibody (hP67.6) and a toxic calicheamicin-γ1 derivative, has proven effective in some patients with AML, but its effectiveness is limited by drug efflux, in particular mediated by P-glycoprotein. GO exerts it toxic effect by calicheamicin-γ1-induced DNA damage, which is followed by either successful DNA repair or cell death. Heat-shock protein 90 (HSP90) interacts with numerous client proteins and guides folding, localization, and turnover of key regulators of cell growth, cell cycle, differentiation, and survival. Inhibition of HSP90, e.g. with 17-allylamino-17-demethoxygeldanamycin (17-AAG), increases degradation of such proteins and can interfere with oncogenic signaling pathways and may increase the sensitivity of tumor cells to DNA damage. In this study, we tested whether 17-AAG sensitizes human AML cell lines and primary AML cells to the cytotoxic effect of GO or calicheamicin-γ1, in vitro, thus potentially overcoming drug resistance.

Methods: CD33+ human AML cell lines and primary blast cells from patients with AML were treated continuously with various concentrations of GO (doses given as calicheamicin dose-equivalent) or free calicheamicin-γ1 in the presence or absence 17-AAG (25–500 nM) for 3 days before cytotoxicity was determined by flow cytometry with propidium iodide (PI). Phosphorylation of histone H2AX as measure for DNA damage was quantified by flow cytometry after 4 hours of incubation with drugs.

Results: Relatively non-toxic doses of 17-AAG significantly enhanced both GO- and calicheamicin-γ1-induced cytotoxicity in cell lines that do not express P-glycoprotein (Pgp), such as ML-1 and NB4 cells (ML-1: increase of PI+ cells from 7.22±1.16% [mean±SEM] to 27.87±3.85% at 0.01 ng/mL GO [p<0.001, n=4] and 5.53±0.12% to 34.05±4.58% at 0.025 ng/mL calicheamicin-γ1 [p<0.001, n=3] vs 2.25±0.09% to 7.59±0.68% with medium only; NB4: increase of PI+ cells from 9.38±1.43% to 27.21±2.38% at 1.0 ng/mL GO [p<0.01, n=4] and 14.37±3.68% to 32.71±3.22% at 0.5 ng/mL calicheamicin-γ1 [p<0.01, n=3] vs 2.09±0.42% to 5.99±1.58% with medium only). Importantly, 17-AAG also significantly increased both GO and calicheamicin-γ1-induced cytotoxicity in Pgp+ KG-1 cells, an effect observed even in the absence of a Pgp inhibitor, indicating that 17-AAG can overcome drug efflux mediated resistance. 17-AAG did not increase the extent of H2AX phosphorylation, suggesting that 17-AAG does not increase the amount of DNA damage but rather sensitizes the cell to the DNA-damage induced by the calicheamicin-γ1 derivative. Finally, 9 samples of primary AML cells, GO (2.5 ng/mL) decreased the fraction of live cells by 19.66±2.77%; 17-AAG at 250 nM decreased the fraction of live cells by 1.10±2.56% when given alone, but decreased the live cell fraction by 36.17±5.56% when given in combination with GO (P<0.0005).

Conclusion: 17-AAG has significant in vitro activity in enhancing cytotoxicity of calicheamicin-based agents, including GO, at clinically achievable concentrations in both Pgp and Pgp+ AML cell lines as well as primary AML cells with limited susceptibility to GO. Therefore, further study of its role as chemosensitizer for GO-based anti-AML therapies seems warranted.

Disclosure: No relevant conflicts of interest to declare.